scFv Antibodies Which Pass Epithelial and/or Endothelial Layers

ABSTRACT

scFv antibodies which specifically bind selected antigens and are obtainable by a method comprising (i) selecting from a pool of soluble and stable antibody frameworks a soluble and stable framework matching best the framework of a non-human antibody against the antigen with a certain binding specificity, (ii) either providing said soluble and stable framework with CDRs that bind specifically to said antigen, or mutating the framework of said non-human antibody towards the sequence of said soluble and stable framework, to generate scFv antibodies, (iii) testing the generated antibody for solubility and stability, and testing the generated antibody for antigen binding, and (iv) selecting an scFV that is soluble, stable and binds to the antigen specifically. Also provided are pharmaceutical compositions comprising said scFv antibody, methods of treatment and diagnosis for diseases related to over expression of antigens that are specifically bound by said antibody.

The present application is a divisional of U.S. application Ser. No.12/307,875, filed on Jul. 30, 2009 (now pending), which is a 371national stage application of International Application No.PCT/CH07/00334, filed on Jul. 10, 2007, which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/899,907, filed on Feb. 6,2007, and U.S. Provisional Patent Application Ser. No. 60/819,378, filedon Jul. 10, 2006, the disclosures of which are specifically incorporatedby reference herein.

FIELD OF THE INVENTION

The present invention concerns an scFv antibody with improved featuresfor tissue penetration and its topical application in the diagnosis andtreatment of a disease dependent on the over-expression of a selectedantigen. In particular, the invention concerns an antibody whichspecifically binds and inactivates said selected antigen.

RELATED INFORMATION

The contents of any patents, patent applications, and references citedthroughout this specification are hereby incorporated by reference intheir entireties.

BACKGROUND OF THE INVENTION

Local treatment of many diseases can occur by topical application of adrug that must be able to penetrate epithelial tissue. Adjacentepithelial cells are sealed by tight junctions, preventing the passageof most dissolved molecules from one side of the epithelial sheet to theother (Alberts et al., Molecular Biology of the Cell, 2nd ed.). Tightjunctions are crucial for the formation and maintenance of theparacellular barrier and for cell polarity in simple epithelia andendothelia. They also play an important role at the blood brain barrier,where they control substances that leave or enter the brain. Largemolecular weight drugs need to pass through these tissue barriers inorder to get to their sites of action. In general, antibodies are toobig to cross the tight junctions of epithelial cell layers.

As part of the body's normal activity, tight junctions selectively openand close in response to various signals both inside and outside ofcells. This allows the passage of large molecules or even entire cellsto across the tight junction barrier.

Mucosal administration of therapeutic compounds may offer certainadvantages over injection and other modes of administration, for examplein terms of convenience and speed of delivery, as well as by reducing oreliminating compliance problems and side effects that attend delivery byinjection. However, mucosal delivery of biologically active agents islimited by mucosal barrier functions and other factors. For thesereasons, mucosal drug administration typically requires larger amountsof drug than administration by injection. Other therapeutic compounds,including large molecule drugs, peptides and proteins, are oftenrefractory to mucosal delivery.

The ability of drugs to permeate mucosal surfaces, unassisted bydelivery-enhancing agents, appears to be related to a number of factors,including molecular size, lipid solubility, and ionization. Smallmolecules, less than about 300-1,000 daltons, are often capable ofpenetrating mucosal barriers, however, as molecular size increases,permeability decreases rapidly. Lipid-soluble compounds are generallymore permeable through mucosal surfaces than are non-lipid-solublemolecules. Peptides and proteins are poorly lipid soluble, and henceexhibit poor absorption characteristics across mucosal surfaces.

US2006062758 provides compositions and methods that include abiologically active agent and a permeabilizing peptide effective toenhance mucosal delivery of the biologically active agent in a mammaliansubject. The permeabilizing peptide reversibly enhances mucosalepithelial paracellular transport, typically by modulating epithelialjunctional structure and/or physiology at a mucosal epithelial surfacein the subject.

Peptides capable of modulating the function of epithelial tightjunctions have been previously described (Johnson, P. H. and Quay, S.C., 2000). CA2379661 provides a paracellular drug delivery systemcomprising a Caludin-6 derived peptide. Claudins represent asuper-family of integral membrane proteins located at the tightjunctions and providing the barrier function.

Antibodies are powerful tools for biochemical and molecular biologyresearch and are widely applied in medical diagnostics and therapy dueto their ability to specifically bind to their antigen with highaffinity. Typically, antibodies consist of two heavy and two lightchains, which are covalently linked to each other via disulfide bonds. Ahighly variable domain comprising three complementary regions (CDRs) ispositioned at the N-terminus of each chain. In concert, variable regionsof the heavy and light chain determine antigen specificity of theantibody. Single chain antibodies (scFv) have been engineered by linkingthe DNA sequences encoding variable heavy (VH) and variable light (VL)domains with a spacer sequence coding for a flexible amino acid linker(Bird et al., 1988).

This format has the advantages over conventional full-length antibodiesthat an scFv is encoded by a single gene, mutations can be easilyintroduced, and the resulting scFv can be expressed in yeast andprokaryotic systems, which allow for rapid selection of specific highaffinity binders to virtually any epitope by simple molecular biology.Due to their lack of effector function, scFv antibodies do not exerttoxic effects via antibody-dependent or complement-dependentcell-mediated cytotoxicity (ADCC or CDCC, respectively), and unlikefull-length antibodies, scFv antibodies show good tissue penetrationabilities.

Many single chain antibodies (scFvs) have been generated against amultitude of different antigens, in particular because they can beeasily selected for high binding capacity using techniques such as phagedisplay or ribosome display. Moreover, scFv antibodies can be producedin microbial systems which are associated with fewer costs compared tothe production of therapeutic full-length antibodies.

In addition to conventional extracellular and in vitro applications,scFvs have also been successfully used for intracellular applications(Worn et al. 2000; Auf der Maur et al. 2002; Stocks M R, 2004); hence,scFvs directed against intracellular antigens have been developed. Ingeneral, intracellular expression of functional scFvs is limited bytheir instability, insolubility, and tendency to form aggregates. Forthis reason, in vivo screening systems for scFv antibodies, which areparticularly soluble and stable under reducing conditions typical forthe intracellular environment (e.g. nucleus, cytoplasm) have beensuccessfully developed using a so called “Quality Control” screen(WO0148017; Auf der Maur et al. (2001); Auf der Maur et al., 2004) andhave led to the identification of particularly stable and soluble scFvframework sequences for such purposes (WO03097697). Furthermore, theseframeworks show exceptional expression levels and enhanced stability andsolubility properties also under natural, oxidizing conditions in theextracellular environment. Hence, these favourable biophysical andbiochemical properties translate into favourable high production yieldsand enable these antibody fragments, once directed against specificantigens, to be applied locally and/or systemically as proteintherapeutics in particular therapeutic areas.

For the use of antibodies in many therapeutic applications, inparticular local applications, an important factor is the ability of theantibody to penetrate tissues, and in particular epithelial tissuebarriers.

Local application is particularly desirable for the treatment ofdisorders that are manifested at a particular locus and do not require asystemic treatment, for example eye diseases.

Uveitis Anterior

Uveitis is an acute or chronic inflammation of the uvea with aprevalence of 30-40 per 100,000 (Lightman and Kok 2002). Uveitis issubdivided by location in uveitis anterior, intermediate or posterior.Uveitis anterior develops into uveitis posterior followed bycomplications such as cataracts, retinitis and even blindness, if leftuntreated (Kok and Lightman 2004). In <65 year olds there are as manylegally blind individuals as a result from uveitis as diabeticretinopathy (Kok and Lightman 2004). Uveitis anterior, as the mostcommon form of intra-ocular inflammatory diseases, is associated withhistocompatibility locus A allele B27 (HLA-B27) in 50% of the cases(Power et al. 1998). Of these patients, only about half suffer from anadditional systemic disease such as ankylosing spondylitis or chronicinflammatory bowel disease (El-Shabrawi and Hermann 2002). The treatmentof uveitis is primarily aimed at controlling the inflammatory process(Kok and Lightman 2004). Currently, corticosteroids are the mainstay fortherapy of uveitis (Kok and Lightman 2004). Importantly, local andsystemic corticosteroid treatment significantly increases the risk ofglaucoma and cataract, thus limiting its repeated use (El-Shabrawi andHermann 2002). Other treatments including methotrexate, cyclosporine orazathioprine require a minimum of 6 weeks treatment to produce aneffect, leaving patients with an enormous constraint of quality of lifefor a long period (El-Shabrawi and Hermann 2002, Dick et al. 1997).

From the above, a clearly defined medical need is obvious. Topicalcorticosteroids as the most common therapeutic option have significantside-effects, which in fact exacerbate the long term risk of blindness.

Recently TNFα concentrations of 15 pg/ml have been found in aqueoushumor of uveitis patients, whereas the corresponding levels in healthyindividuals were 0.56 pg/ml (Perez-Guijo et al. 2004). Several smallclinical studies performed with systemically applied TNFα inhibitorsreport “immediate improvement” (El-Shabrawi and Hermann 2002) or “markedclinical improvement within days” (Murphy et al. 2004) or “within 2weeks” (Joseph 2003) or “significant improvement after the firstinfliximab dose” (Benitez Del Castillo et al. 2004).

Thus, the concept of targeting TNFα is clinically well validated.However, safety concerns related to systemic application of TNFαinhibitors remain and would not justify their use in the significantfraction of uveitis patients who lack additional systemic diseasemanifestations.

Therefore, a topical TNFα inhibitor will fill a well-defined medicalneed, especially in patients with uveitis anterior. Due to their largemolecular weight, the marketed TNFα inhibitors are not applicabletopically (see Thiel et al. 2002).

Behçet's Disease

Behçet's disease is an idiopathic, multisystemic, chronic, and recurrentdisease, classically characterized by episodic aggressive ocularinflammatory attacks, orogenital ulcers and skin lesions. In rare,severe cases of Behçet's disease, articular, audio-vestibular, thoracicgastrointestinal, cardiovascular, renal or CNS involvement may beobserved in addition. The eye is the most commonly involved internalorgan in Behçet's disease and is the leading cause of chronic morbidityin patients. Ocular disease consists of unilateral (20%) or bilateral(80%) iridocyclitis, hypopyon or panuveitis running a chronic andrelapsing course. In general, initial exacerbations tend to be moreanterior and unilateral, whereas subsequent attacks tend to involvevitreal cavity and posterior segment of the eye, becoming bilateral(Evereklioglu 2005). Severe uveitis is more commonly observed amongpatients form endemic regions such as Japanese and Turkish patients,affecting 70-90% of this population (Özen 1999; Tursen et al., 2003;Tugal-Tutkun et al., 2004; Yurdakul et al., 2004; Evereklioglu 2005).The risk of visual loss increases progressively, reaching one-fourth ofthe cases at 10 years. In addition, legal blindness is significant andeventually ensues in more than 50% of cases in countries with highprevalence and severity of the disease, such as Japan (Boyd et al.,2001; Evereklioglu 2005).

Behçet's disease exhibits a distinct geographic variation and isendemically higher particularly in Japan, Korea, Saudi-Arabia, Iran andTurkey as well as in the countries along the ancient “silk road”,including China and Israel (Bonfioli and Orefice 2005; Evereklioglu2005). For example, Behçet's disease accounts for 20% of cases ofuveitis in Japan and Turkey when compared with only 0.2% in the USA. Incountries where the disease is endemic, it is more severe, with a higherfrequency of ocular manifestations and complications and is more commonin men, especially young male adults (Evereklioglu 2005). This peculiarepidemiology appears to be mediated by a combination of genetics (suchas association with the HLA-B51 allele (Sakane et al., 1999; Verity etal., 1999; Evereklioglu 2005), infectious agents (Direskeneli 2001;Evereklioglu 2005) and environmental factors. The estimated prevalenceof Behçet's disease is between 1:10,000 and 1:1000 in the Mediterraneancountries, the Middle East, and the Far East. In Japan and Asiancountries along the silk road, the prevalence is 13-30 per 100,000 andis highest in the northern parts of Japan; the highest overallprevalence with up to 400 per 100,000 is observed in certain parts ofTurkey. There are approximately 15,000 people with Behçet's disease inthe USA (Zierhut et al., 2003; Evereklioglu 2005).

Consequences of ophthalmic inflammatory attacks are the leading cause ofchronic morbidity in Behçet's disease patients (Evereklioglu 2005).Treatment of Behçet's disease is symptomatic and empirical. As in otherforms of uveitis, topical, periocular and systemic corticosteroidsrepresent the mainstay of therapy in ocular Behçet's disease. However,the use of corticosteroid-based treatment modalities in the patients islimited by their significant side-effect profile. In addition,corticosteroids rarely induce complete remissions in ocular Behçet'sdisease and a significant fraction of patients developssteroid-resistant disease over time (Evereklioglu 2005). In the courseof the disease, treatments frequently comprise immunosuppressive agentssuch as azathioprine, methotrexate and cyclosporine A. However, as theseagents are associated with critical safety issues as well, there is awell-expressed medical need for an efficient and safe novel treatmentmodality in this indication.

Besides recent epidemiological findings that suggest polymorphicvariations in TNFα to be associated with the severity of Behçet'sdisease (Verity et al., 1999b), there exists a broad variety of casereports and small clinical trials describing the use of infliximab inocular Behçet's disease (Ohno et al., 2004; Wechsler et al., 2004;Giansanti et al., 2004; Lanthier et al., 2005; Tugal-Tutkun et al.,2005; Lindstedt et al., 2005). In fact, all these studies report rapidand complete remission of ocular Behçet's disease, even in patientsresistant to conventional therapy (Tugal-Tutkun et al., 2005). However,the frequency and severity of adverse events in infliximab-treateduveitis patients is unexpectedly high in some studies, thus limiting thepotential of systemically applied TNFα antagonists for treatment of thisdisease (Rosenbaum 2004; Suhler et al., 2005).

The clinical validation of TNFα as a highly attractive drug target inocular Behçet's disease (Ohno et al., 2004; Wechsler et al., 2004;Giansanti et al., 2004; Lanthier et al., 2005; Tugal-Tutkun et al.,2005; Lindstedt et al., 2005) and the apparent safety concerns withsystemic TNFα suppression in uveitis patients (Rosenbaum 2004; Suhler etal., 2005), reveals that there is a need for the development of atopically applicable TNFα antagonist for ocular Behçet's disease,especially for patients with predominant ocular symptoms.

Due to their good tissue penetration abilities and their rapid renalclearance, scFv antibodies are preferred for local applications. Besidescharge, hydropathicity and molecular weight, properties such assolubility, aggregation tendency and thermal stability influence theability of a molecule to penetrate through tissue barriers. For example,a highly soluble antibody fragment may not be able to penetrateepithelial barriers if it forms aggregates at physiological temperaturearound 37° C. Mutation of a single amino acid residue in an scFvframework may on the one hand improve its solubility at ambienttemperature, and this mutation may alter thermal stability and thereforelead to partial unfolding and aggregation at 37° C. Such aggregates, dueto their higher molecular weight, are no longer able to pass tissuebarriers.

Because tissue penetration is an important factor for efficient drugdelivery, in particular in local applications, there is a need fortherapeutic antibodies, in particular scFv antibodies with improvedtissue penetration abilities besides the otherwise desirablecharacteristics of high stability and low antigenicity. WO0040262discloses antibody fragments, e.g. scFvs, as pharmaceuticals ordiagnostic tools to treat or diagnose, respectively, ocular disorders.Eye penetration experiments are done at concentrations of 0.2 to 0.25mg/ml scFv. It was shown that an scFv could penetrate the epithelialbarrier of the cornea at a very low rate in the absence, and at higherrates in the presence of penetration enhancers. Since penetrationenhancers can have cytotoxic effects or cause epithelial alterations,there is a need for alternative and/or improved methods for thetreatment of ocular diseases by scFvs and fragments thereof. Inparticular, antibodies are needed for controlled therapy by localadministration with a low degree of side effects, which can beadministered at a relatively high concentration.

All publications and references cited herein are hereby incorporated byreference in their entirety.

SUMMARY OF THE INVENTION

Hence, it is a general object of the invention to provide an antibody,preferably an scFv antibody, which specifically binds a selected antigenand has improved tissue penetration ability.

Now, in order to implement these and still further objects of theinvention, which will become more readily apparent as the descriptionproceeds, said antibody is manifested by the feature that it isobtainable by a method comprising

(i) selecting from a pool of soluble and stable frameworks the frameworkmatching best to the framework of a non-human antibody of a selectedantigen-binding specificity,

(ii) either providing said framework with CDRs that bind said antigen ormutating the framework of said non-human antibody towards the sequenceof said soluble and stable framework,

(iii) testing the generated antibody for solubility and stability, and

(iv) testing the generated antibody for antigen binding.

Optionally, between steps (ii) and (iii) the following step is added:

-   -   mutating said scFv antibody by site-directed or random        mutagenesis of one or more selected CDRs and/or the framework.

The invention also provides a composition comprising a solubleantigen-binding polypeptide, wherein the antigen-binding polypeptide iscapable of crossing one or more epithelial layers, for example, anendothelial layer or mesothelial layer, in less than about 8 hours. Forexample, the antigen-binding polypeptide is capable of crossing one ormore epithelial layers in less than about 8, 7, 6, 5, 4, 3, 2, 1, orfewer hours. In one embodiment, the antigen-binding polypeptide iscapable of crossing an epithelial layer or layers in less than about 4hours. It is to be understood that all values and ranges between thesevalues and ranges are meant to be encompassed by the present invention.

In other embodiments, the epithelial layer is of the eye, for example,of the cornea, for example the epithelium and/or endothelium of thecornea. In one embodiment, the epithelial layer is of the intestine. Inyet another embodiment, the epithelial layer is the blood brain barrier.

In yet other embodiments, the antigen-binding polypeptide is capable ofcrossing an intact mammalian cornea in less than about 8 hours. Forexample, the antigen-binding polypeptide is capable of crossing anintact mammalian cornea in less than about 8, 7, 6, 5, 4, 3, 2, 1 orfewer hours. In one embodiment, the antigen-binding polypeptide iscapable of crossing an intact human cornea. In one embodiment, theantigen-binding polypeptide is capable of crossing an intact pig orrabbit cornea.

In still other embodiments, the composition further comprises apenetration enhancer. In certain embodiments, the penetration enhanceris selected from the group consisting of Azone®, benzalkonium chloride(BzCl), BL-7, BL-9, Brij 35, Brij 78, Brij 98, Brij 99,Polyoxyethylene-Polyoxypropylene 1800, sodium caprate, caprylic acid,cetylpyridinium chloride, chlorhexidine, cholate, castor oil, corn oil,cremophor-EL, cyclodextrins, DMSO, decamethonium bromide, deoxycholate,dextransulfate, EDTA, disodium EDETATE, ethanol, fusidate, glycocholate,lauryl sulfate, L-α-lysophosphatidylocholine, methazolamide,N-lauroylsarcosine, NMP, oleic acid, Pz-peptide, phospholipids, polyoxyethylene-9-lauryl ether, saponin, Tween 20, Tween 40, Tween 60, Tween80, taurocholeate, and taurodeoxycholate. In another embodiment, thepenetration enhancer is sodium caprate. In yet another embodiment, thepenetration enhancer includes colloidal systems, polyacrylates andbio-adhesive polymer.

In a preferred embodiment, the molecules of the invention can cross anepithelial layer, for example, an epithelial layer of the eye (cornea)in the absence of a penetration enhancer.

In some embodiments, the polypeptide has a binding affinity for a targetantigen of a kD of at least 10E-6 M or better.

In some aspects, the present invention provides a composition having apH of less than about 8, said composition comprising an antigen-bindingpolypeptide (e.g., single-chain antibody) wherein the polypeptide issufficiently soluble to transit an intact cornea. In some embodiments,the composition has a pH in the range of about 6 to about 8. In otherembodiments, the composition has a pH of about 6, 6.5, 7.0, 7.5, 8.0 orany incremental value thereof. It is understood that any values andranges between these values and ranges are meant to be encompassed bythe present invention.

In some aspects, the present invention provides a composition having apH of less than about 8, said compositions comprising an antigen-bindingpolypeptide (e.g., single-chain antibody) wherein the polypeptide issufficiently soluble to transit an intact cornea. In some aspects, thepresent invention provides a composition comprising a solubleantigen-binding polypeptide, and wherein the polypeptide is sufficientlysoluble to transit an intact cornea in less than about 8 hours, andformulated at about pH 8 or less. In some embodiments, the polypeptideis sufficiently soluble to transit an intact cornea in less than about 4hours. In other embodiments, the composition further comprises apenetration enhancing agent. In some embodiments, the penetrationenhancing agent is selected from the group consisting of azone,benzalkonium chloride (BzCl), BL-7, BL-9, Brij 35, Brij 78, Brij 98,Brij 99, Polyoxyethylene-Polyoxypropylene 1800, sodium caprate, caprylicacid, cetylpyridinium chloride, chlorhexidine, cholate, castor oil, cornoil, cremophor-EL, DMSO, decamethonium bromide, deoxycholate,dextransulfate, EDTA, disodium EDETATE, ethanol, fusidate, glycocholate,lauryl sulfate, L-α-lysophosphatidylocholine, N-lauroylsarcosine, NMP,oleic acid, phospholipids, poly oxyethylene-9-lauryl ether, saponin,Tween 20, Tween 40, Tween 60, Tween 80, taurocholeate, andtaurodeoxycholate. In some embodiments, the penetration enhancing agentis sodium caprate. In some embodiments, the penetration enhancing agentis chlorhexidine.

In some aspects, the present invention provides a composition comprisinga soluble antigen-binding polypeptide, wherein the antigen-bindingpolypeptide is capable of crossing one or more layers of an intactcornea in less than about 8 hours. In other aspects, the presentinvention provides a composition comprising an antigen-bindingpolypeptide (e.g., single-chain antibody) at a concentration of greaterthan about 2.5 mg/ml, wherein the polypeptide is sufficiently soluble totransit an intact cornea in less than about 8 hours. The composition cancomprise an antigen-binding polypeptide at a concentration in the rangeof from greater than about 2.5 mg/ml to greater than about 10.0 mg/ml.For example, the composition can comprise an antigen-binding polypeptideat a concentration of about 2.5 mg/ml, 3.0 mg/ml, 3.5 mg/ml, 4.0 mg/ml,4.5 mg/ml, 5.0 mg/ml, 5.5 mg/ml, 6.0 mg/ml, 6.5 mg/ml, 7.0 mg/ml, 7.5mg/ml, 8.0 mg/ml, 8.5 mg/ml, 9.0 mg/ml, 9.5 mg/ml, to greater than about10.0 mg/ml, or any incremental value thereof. It is to be understoodthat all values and ranges between these values and ranges are meant tobe encompassed by the present invention. In some embodiments, theantigen-binding polypeptide is at a concentration of greater than about4.0 mg/ml. In other embodiments, the antigen-binding polypeptide is at aconcentration of greater than about 10.0 mg/ml.

In yet other embodiments, the polypeptide is sufficiently soluble totransit an intact cornea in less than about 4 hours. In otherembodiments, the composition further comprises a penetration enhancingagent. In some embodiments, the penetration enhancing agent is selectedfrom the group consisting of azone, benzalkonium chloride (BzCl), BL-7,BL-9, Brij 35, Brij 78, Brij 98, Brij 99,Polyoxyethylene-Polyoxypropylene 1800, sodium caprate, caprylic acid,cetylpyridinium chloride, chlorhexidine, cholate, castor oil, corn oil,cremophor-EL, DMSO, decamethonium bromide, deoxycholate, dextransulfate,EDTA, disodium EDETATE, ethanol, fusidate, glycocholate, lauryl sulfate,L-α-lysophosphatidylocholine, N-lauroylsarcosine, NMP, oleic acid,phospholipids, poly oxyethylene-9-lauryl ether, saponin, Tween 20, Tween40, Tween 60, Tween 80, taurocholeate, and taurodeoxycholate. In someembodiments, the penetration enhancing agent is sodium caprate. In otherembodiments, the penetration enhancing agent is chlorhexidine.

In some aspects, the present invention provides an antigen-bindingpolypeptide (e.g., single-chain antibody), having a binding affinity fora target antigen of a kD of at least 10E-6 M and wherein the polypeptideis sufficiently soluble to transit an epithelial tight junction, andwherein the polypeptide remains in monomeric form under physiologicalconditions.

In other aspects, the present invention comprises a compositioncomprising an antigen-binding polypeptide wherein the antigen-bindingpolypeptide is stable at a temperature from about −80 degrees Celsius toabout 37 degrees Celsius. For example, the composition may be stable ata temperature of −80 degrees Celsius, −70 degrees Celsius, −60 degreesCelsius, −50 degrees Celsius, −40 degrees Celsius, −30 degrees Celsius,−20 degrees Celsius, −10 degrees Celsius, 0 degrees Celsius, 10 degreesCelsius, 20 degrees Celsius, or 30 degrees Celsius, or any incrementalvalue thereof. It is to be understood that all values and ranges betweenthese values and ranges are meant to be encompassed by the presentinvention. In some embodiments, the antigen-binding polypeptide remainsstable for at least about eight weeks. In other embodiments, theantigen-binding polypeptide remains stable for at least six weeks at 4degrees Celsius.

In some aspects, the present invention provides a composition comprisingan antigen-binding polypeptide, wherein the antigen-binding polypeptidehas the pharmacodynamic or pharmacokinetic features as experimentallyshown throughout any of the figures disclosed herein.

In other aspects, the present invention provides an antigen-bindingpolypeptide having a binding affinity for a target antigen of a kD of atleast 10E-6 M or better and wherein the polypeptide is sufficientlysoluble to transit an epithelial tight junction in less than about 8hours. In some embodiments, the polypeptide is sufficiently soluble totransit an epithelial tight junction in about 4 hours or less.

In still yet other aspects, the present invention provides anantigen-binding polypeptide having a binding affinity for a targetantigen of a kD of at least 10E-6 M or better and wherein thepolypeptide has a ½ Vmax value corresponding to the transit kinetics ofan antigen-binding polypeptide that can cross an epithelial tightjunction in less than about 8 hours.

In other aspects, the present invention includes an antigen-bindingpolypeptide sufficiently soluble to transit an epithelial tight junctionas measured in a standard Caco-2 (human colon adenocarcinoma) epithelialcell monolayer assay, as to be suitable for use in therapy. In variousaspects, the present invention includes an antigen-binding polypeptidesufficiently soluble to transit an epithelial tight junction as measuredin a standard mouse jejunum permeability assay, as to be suitable foruse in therapy. In yet other aspects, the present invention includes anantigen-binding polypeptide sufficiently soluble to transit anepithelial tight junction as predicted by a standard intracellular onehybrid or two hybrid solubility assays, as to be suitable for use intherapy. In still yet other aspects, the present invention provides anantigen-binding polypeptide sufficiently soluble to transit anepithelial tight junction as predicted by a standard PEG precipitationassay or self-interaction chromatography (SIC) assay, as to be suitablefor use in therapy.

In other aspects the present invention provides a method for identifyingan antigen-binding polypeptide having a ½ Vmax value corresponding totransit of the antigen-binding polypeptide across an epithelial tightjunction in less than about 8 hours. The method comprises: expressingintracellularly candidate antigen-binding polypeptides in host cellshaving an inducible reporter gene system, wherein the reporter genesystem yields a recordable signal when in the presence of anantigen-binding polypeptide having said transit kinetics; and screeningsaid cells for a recordable signal, wherein the presence of said signalidentifies a candidate polypeptide as an antigen-binding polypeptidehaving said transit kinetics. The present invention, in some aspects,also provides for am antigen-binding polypeptide identified by thismethod. In some aspects, the present invention also provides a kit forcarrying out this method.

In other aspects, the present invention provides a method of treating apatient with an ocular condition by topically administering atherapeutically effective amount of an antigen-binding polypeptide ofany one of the claims herein, such that treatment is achieved. In someembodiments, the ocular condition is uveitis. In other embodiments, theocular condition is age related macular degeneration.

In some aspects, the present invention provides an antigen-bindingpolypeptide comprising a polypeptide region having at least oneantigen-binding motif flanked by at least one scaffold region andwherein the polypeptide has transit kinetics sufficient to cross anepithelial tight junction in less than about 8 hours. In someembodiments, the polypeptide comprises one antigen-binding motif flankedby two scaffold regions, two antigen-binding motifs flanked by threescaffold regions, three antigen-binding motifs flanked by four scaffoldregions, or six antigen-binding motifs flanked by eight scaffold regionswith an intervening linker region between the fourth and fifth scaffoldregions. In other embodiments, the antigen-binding motif is a CDR andthe scaffold region is an immunoglobulin framework region. In still yetother embodiments, the polypeptide comprises three CDRs and fourintervening framework regions or six CDRs and eight framework regionsand an intervening linker region.

In some aspects, the present invention also provides an antigen-bindingpolypeptide capable of specifically binding a target antigen and havingtransit kinetics sufficient to cross an epithelial tight junction inless than about 8 hours and wherein the polypeptide is represented bythe formula:

Y; or

Z; or

Y-L-Z; or

Z-L-Y;

with Y being [F1-CDR1-F2-CDR2-F3-CDR3 F4] and Z being[F5-CDR1-F6-CDR2-F7-CDR3F8];

wherein the framework regions (F1-F4) of Y are derived from one or morehuman light chain frameworks; the framework regions (F5-F6) of Z arederived from one or more human light chain frameworks; the CDRs(CDRs1-3) of Y are derived from one or more donor CDRs capable ofbinding the target antigen; the CDRs (CDRs4-6) of Z are derived from oneor more donor CDRs capable of binding the target antigen; and L is aflexible polypeptide linker. In some embodiments, Y and Z arerepresented by any of the sequences disclosed herein, or consensusthereof.

Alternatively, in another embodiment, mutations can be are randomlyintroduced randomly along all or part of the antigen-binding polypeptidecoding sequence, such as by saturation mutagenesis. A “consensussequence” is a sequence formed from the most frequently occurring aminoacids (or nucleotides) in a family of related sequences (See e.g.,Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany1987). In a family of proteins, each position in the consensus sequenceis occupied by the amino acid occurring most frequently at that positionin the family. If two amino acids occur equally frequently, either canbe included in the consensus sequence.

In some aspects, the present invention provides an antigen-bindingpolypeptide formulated to achieve an intraocular concentration of atleast about 100 ng/ml or more. In yet other aspects, the presentinvention provides a single chain antibody formulated for topicaladministration to yield an intraocular concentration of 100 ng/ml ormore based on a cellular or animal model system as disclosed herein.

In other aspects, the present invention provides an antigen-bindingpolypeptide formulated for topical application to eye and capable ofpassing through the cornea and into an intraocular space in the absenceof penetration enhancer. In yet other aspects, the present inventionprovides a method for treating, preventing or diagnosing an eye diseaseor disorder using a polypeptide of any one of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other aspects, embodiments, objects, features andadvantages of the invention can be more fully understood from thefollowing description in conjunction with the accompanying drawings. Inthe drawings like reference characters generally refer to like featuresand structural elements throughout the various figures. The drawings arenot necessarily to scale, emphasis instead being placed uponillustrating the principles of the invention.

FIG. 1 shows the elution profiles of ESBA105 (FIG. 1A), TB-WT (FIG. 1B)and lucentis-scFv (FIG. 1C) of a preparative size exclusionchromatography (gel filtration) following the refolding step. mAU:milli-absorbance units.

FIG. 2 shows the elution profile of an analytical gel filtration of thecollected peak fractions of ESBA105 after preparative gel filtration.

FIG. 3 shows the solubility of ESBA105 in polyethylene glycol (PEG).

FIG. 4 shows the stability of ESBA105 and QC 15.2 scFv antibodies whenstored for two weeks at different temperatures and concentrations.Antibodies were separated by SDS PAGE and stained with Coomassiebrilliant blue.

FIG. 5 shows the activity of ESBA105 determined by an L929 assay after 8weeks of storage at either 37° C. or −80° C., both at pH 7.4. Trianglesindicate ESBA105 stored at 37° C. and squares ESBA105 stored at −80° C.

FIG. 6 shows schematically syringes removing liquid from vitreous andfrom anterior chamber, respectively. 1 vitreous cavity liquid, 2anterior eye liquid, 3 iris, 4 cornea, 13 syringe.

FIG. 7 shows penetration of ESBA105 into the anterior chamber of intactrabbit eyes after 4 hours.

FIG. 8 shows penetration of ESBA105 the vitreous cavity of intact rabbiteyes after 4 hours.

FIG. 9 shows penetration of ESBA105 across a Caco-2 cell layer.

FIG. 10 shows a comparison of penetration efficacies of a full-lengthIgG format antibody (Infliximab) and a single-chain format antibodyfragment (ESBA105) through rat jejunum in the non-everted sac model forintestinal drug absorption. Black squares indicate the ESBA105concentration in nM, white circles the infliximab concentration in nM.

FIG. 11 a is a graphical depiction of the amount of ESBA105 in ng/mlfound in the aqueous humour of the rabbit eyes sampled over the courseof the study described in Example 7.

FIG. 11 b is a graphical depiction of the amount of ESBA105 in ng/mlfound in the vitreous humour of the rabbit eyes sampled over the courseof the study described in Example 7.

FIG. 11 c is a graphical depiction of the amount of ESBA105 in ng/mlfound in the neuroretina of the rabbit eyes sampled over the course ofthe study described in Example 7.

FIG. 11 d is a graphical depiction of the amount of ESBA105 in ng/mlfound in the serum of the rabbit eyes sampled over the course of thestudy described in Example 7.

FIG. 12 is a graphical depiction of the local in vitro pK of ESBA105 inrabbit eyes. Retina extract ˜500 ng/ml. 060721 ELISA (from #060718 WholeEye Rabbit), Retina.

FIG. 13 is a graphical depiction of the local half-time of ESBA105 uponvitreal injection in rabbit eyes.

FIG. 14 is a graphically depicts the modeling of local drug accumulationafter administration of ESBA105 (5 drops/day, 10 mg/ml ESBA105,P_(elf)=2.9×10⁻⁵).

FIG. 15 is a graphical depiction of the pK of the eye. 4 cornea, 5 tearfilm, 6 anterior chamber, 7 lens, 8 vitrous body, 9 retina, 10 sclera.

FIG. 16 is a graphical depiction of absorption and elimination routesfor ESBA105. 11 hydrophilic drugs, 12 lipophilic drugs.

FIG. 17 shows dose response data in a relevant in vivo acutemonoarthritis (rat) model. n=3, TNFα: 10 μg i.a.

FIG. 18 A shows a graphical depiction of topical rabbit eye applicationin vivo results. Each data point represents the average of two rabbits(four eyes), which received one drop (30 mcl) of 10 mg/ml ESBA105 in PBSpH 6.5 solution every 20 minutes over a maximal treatment period of 10hours.

The drops were applied on the top of the pupil and the eyelids weresubsequently squeezed to remove excess fluid (7 mcl remaining). ESBA105concentrations were determined in aqueous, vitreous and serum by ELISA.

FIG. 19 shows a graphical depiction of topical rabbit eye application invivo results. One drop of 10 mg/ml ESBA105 solution was applied to thelower eye sac of both eyes of each animal five times a day for up to 6days.

Sampling: After applying the second drop at the indicated time point(after 1, 3 or 6 days) two animals were sacrificed and both eyes, aswell as the serum were subjected to quantitative ELISA analysis. ESBA105levels in were determined in the aqueous (FIG. 19A), in the vitreous(FIG. 19 B), in the neuroretina (FIG. 19 C), in the choroidea (FIG. 19D) and in the serum (FIG. 19 E) as indicated.

“Carr” stands for carrier, which means buffer solution without ESBA105.Data bars represent the maximal, the minimal and the median ESBA105concentrations measured in the indicated compartments and are giventogether with the respective standard deviations.

DETAILED DESCRIPTION OF THE INVENTION

In order to provide a clear understanding of the specification andclaims, the following definitions are conveniently provided below.

DEFINITIONS

The term “antibody” refers to whole antibodies and any antigen-bindingfragment (i.e., “antigen-binding portion,” “antigen-bindingpolypeptide,” or “immuno-binder”) or single chain thereof. An “antibody”refers to a glycoprotein comprising at least two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, or anantigen-binding portion thereof. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as VH) and a heavy chainconstant region. The heavy chain constant region is comprised of threedomains, CH1, CH2 and CH3. Each light chain is comprised of a lightchain variable region (abbreviated herein as VL) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL. The VH and VL regions can be further subdivided into regionsof hypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

The term “antigen-binding” refers to the ability to specifically bind toan antigen. It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a singledomain or dAb fragment (Ward et al., (1989) Nature 341:544-546), whichconsists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR) or (vii) a combination of two or more isolatedCDRs which may optionally be joined by a synthetic linker. Furthermore,although the two domains of the Fv fragment, VL and VH, are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules (knownas single chain Fv (scFv); see e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). These antibody fragments are obtained using conventionaltechniques known to those with skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

As used, “immunoglobulin” may refer to any recognized class or subclassof imununoglobulins such as IgG, IgA, IgM, IgD, or IgE. Theimmunoglobulin can be derived from any species, such as human, murine,or rabbit origin. Further, the immunoglobulin may be polyclonal,monoclonal, or fragments. Such immunoglobulin fragments may include, forexample, the Fab′, F(ab′)2, Fv or Fab fragments, or other antigenrecognizing immunoglobulin fragments. Such immunoglobulin fragments canbe prepared, for example, by proteolytic enzyme digestion, for example,by pepsin or papain digestion, reductive alkylation, or recombinanttechniques. The materials and methods for preparing such immunoglobulinfragments are well-known to those skilled in the art (Parham, (1983) J.Immunology, 131:2895; Lamoyi et al., (1983) J. Immunological Methods,56:235; Parham, (1982) J. Immunological Methods, 53:133; and Matthew etal., (1982) J. Immunological Methods, 50:239).

In addition the immunoglobulin may be a single chain antibody (“SCA”).These may consist of single chain Fv fragments (“scFv”) in which thevariable light (“VL”) and variable heavy (“VH”) domains are linked by apeptide bridge or by disulfide bonds. Also, the immunoglobulin mayconsist of single VH domains (dabs) which possess antigen-bindingactivity. See, e.g., G. Winter and C. Milstein, Nature, 349, 295 (1991);R. Glockshuber et al., Biochemistry 29, 1362 (1990); and, E. S. Ward etal., Nature 341, 544 (1989).

As used herein, the term “polypeptide” refers to a polymer of two ormore of the natural amino acids or non-natural amino acids. Thepolypeptides of the invention comprise at least one amino acid sequencederived from an immunoglobulin (Ig) molecule. In one embodiment apolypeptide of the invention comprises an amino acid sequence or one ormore moieties not derived from an immunoglobulin molecule. Exemplarymodifications are described in more detail below. For example, in oneembodiment, a polypeptide of the invention may comprise a flexiblelinker sequence. In another embodiment, a polypeptide may be modified toadd a functional moiety (e.g., PEG, a drug, or a label).

Preferred polypeptides of the invention comprise an amino acid sequencederived from a human immunoglobulin sequence. However, polypeptides maycomprise one or more amino acids from another mammalian species. Forexample, a primate heavy chain portion, hinge portion, or binding sitemay be included in the subject polypeptides. Alternatively, one or moremurine amino acids may be present in a polypeptide. Preferredpolypeptides of the invention are not immunogenic.

It will also be understood by one of ordinary skill in the art that thepolypeptides of the invention may be altered such that they vary inamino acid sequence from the naturally occurring or native polypeptidefrom which they were derived, while retaining the desirable activity ofnative polypeptide. For example, nucleotide or amino acid substitutionsleading to conservative substitutions or changes at “non-essential”amino acid residues may be made. An isolated nucleic acid moleculeencoding a non-natural variant of a polypeptide derived from animmunoglobulin (e.g., an immunoglobulin heavy chain portion or lightchain portion) can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofthe immunoglobulin such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations may be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis.

The polypeptides of the invention may comprise conservative amino acidsubstitutions at one or more non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in polypeptide is preferably replaced with another amino acidresidue from the same side chain family. In another embodiment, a stringof amino acids can be replaced with a structurally similar string thatdiffers in order and/or composition of side chain family members.Alternatively, in another embodiment, mutations may be introducedrandomly along all or part of the immunoglobulin coding sequence, suchas by saturation mutagenesis, and the resultant mutants can beincorporated into polypeptides of the invention and screened for theirability to bind to the desired target.

The terms “specific binding,” “selective binding,” “selectively binds,”and “specifically binds,” refer to antibody binding to an epitope on apredetermined antigen. Typically, the antibody binds with an affinity(KD) of approximately less than 10-6 M, such as approximately less than10-7 M, 10-8 M or 10-9 M or even lower.The term “KD” refers to the dissociation equilibrium constant of aparticular antibody-antigen interaction. Typically, the antibodies ofthe invention bind to their target antigen with a dissociationequilibrium constant (KD) of less than approximately 10-6 M, such asless than approximately 10-7 M, 10-8 M or 10-9 M or even lower, forexample, as determined using surface plasmon resonance (SPR) technologyin a BIACORE instrument.

The term “transit kinetics” or “pharmacokinetics” as used herein refersto all factors related to the dynamics of drug absorption, distributionin body tissues or fluids and metabolism and/or elimination. Thisinvolves the physicochemical factors that regulate the transfer of thepolypeptide across membranes because the absorption, distribution,biotransformation and excretion of a polypeptide all involve the passageof the polypeptide across cell membranes.

Of great interest to the clinician is the bioavailability of apolypeptide. This term, as used herein, indicates the extent to which apolypeptide reaches its site of action or a biological fluid from whichthe polypeptide has access to its site of action. The factors affectingbioavailability include rate of absorption and metabolism or eliminationof the polypeptide from the subject. Many factors affect absorption,these include the numerous physicochemical factors that affect transportacross membranes such as a polypeptide solubility and uptake mechanismsas well as factors such as site of administration and the formulation(concentration) and composition of the polypeptide. The various routesof polypeptide administration have markedly different absorptioncharacteristics. These routes include oral ingestion, pulmonaryabsorption, parenteral injection, including; intramuscular,subcutaneous, intravenous, intraarterial, intrathecal or intraperitonealinjection and topical application to mucous membranes, skin or eye. In apreferred embodiment, a polypeptide of the invention for treating eyedisease is administered topically to the surface of the eye, e.g., inthe form of eye drops. The transit kinetics of a polypeptide of theinvention can be determined using, for example, any of the cell oranimal based molecules disclosed herein, and are typically selected tobe suitable for clinically relevant transit across the tight junctionsof the blood brain barrier, intestine, or the eye.

The term “subject” is known in the art, and, as used herein, refers to awarm-blooded animal, more preferably a mammal, including, e.g.,non-human animals such as rats, mice, rabbits, cats, dogs, sheep,horses, cattle, in addition to humans. In a preferred embodiment, thesubject is a human. The subjects are those susceptible to treatment witha soluble antigen-binding polypeptide of the present invention.

A “penetration enhancing agent” or “penetration enhancer” as usedherein, refers to molecules or compounds that promote transit across anepithelial junction. Penetration enhancing agents for use with thepresent invention include, but are not limited to, azone, benzalkoniumchloride (BzCl), BL-7, BL-9, Brij 35, Brij 78, Brij 98, Brij 99,Polyoxyethylene-Polyoxypropylene 1800, sodium caprate, caprylic acid,cetylpyridinium chloride, chlorhexidine, cholate, castor oil, corn oil,cremophor-EL, DMSO, decamethonium bromide, deoxycholate, dextransulfate,EDTA, disodium EDETATE, ethanol, fusidate, glycocholate, lauryl sulfate,L-α-lysophosphatidylocholine, N-lauroylsarcosine, NMP, oleic acid,phospholipids, poly oxyethylene-9-lauryl ether, saponin, Tween 20, Tween40, Tween 60, Tween 80, taurocholeate, and taurodeoxycholate. Forexample, in some embodiments, the penetration enhancing agent is sodiumcaprate. In other embodiments, the penetration enhancing agent ischlorhexidine.

In a first aspect of the present invention an scFv antibody is providedthat specifically binds a selected antigen and has improved tissuepenetration ability. Said antibody is characterized in that it isobtainable by a method comprising

(i) selecting from a pool of soluble and stable frameworks the frameworkmatching best to the framework with a non-human antibody of a selectedantigen-binding specificity,

(ii) either providing said framework with CDRs that bind said antigen ormutating the framework of said non-human antibody towards the sequenceof said soluble and stable framework,

(iii) testing the generated antibody for solubility and stability, and

(iv) testing the generated antibody for antigen binding.

The term “matching best” means being as close as possible with respectto primary or tertiary structure.

In general, the antibody of the present invention comprises a frameworkwith a VL and/or a VH domain, said framework being selected from atleast part of a natural human antibody repertoire by an antigenindependent method for high intracellular stability and solubility in ayeast cell. Said method is also known as the “quality control” screeningof antibody frameworks and has resulted in a selection of particularlystable and soluble antibody frameworks that are characterized by highintracellular stability and solubility. These frameworks can be used forexample in a second, yeast based screening system for antigenspecificity. In this case, CDRs of a particularly stable and solubleantibody can be randomized and the resulting antibodies can be screenedfor best possible antigen recognition. Alternatively, known antibodyCDRs of antibodies with strong binding affinity to an antigen of choicecan be grafted onto said particularly stable and soluble frameworks.Optionally, said antibody can be further improved by mutagenesis ofselected CDR(s) and/or framework, selecting improved clones in the“quality control system” (WO0148017, Auf der Maur et al. 2004), i.e. bymutating said scFv antibody by site-directed or random mutagenesis ofone or more selected CDRs and/or the framework and selecting for stableand soluble antibodies under the same or under more stringentconditions. Selection can be done in vivo in the yeast quality controlsystem.

The term “framework residues” relates to amino acid residues ofantigen-binding polypeptide units, or the corresponding amino acidresidues of antigen-binding polypeptide modules, which contribute to thefolding topology, i.e. which contribute to the fold of said unit (ormodule) or which contribute to the interaction with a neighboring unit(or module). Such contribution might be the interaction with otherresidues in the unit (or module), or the influence on the polypeptidebackbone conformation as found in α-helices or β-sheets, or amino acidstretches forming linear polypeptides or loops. The term “targetinteraction residues” refers to amino acid residues of the units, or thecorresponding amino acid residues of the modules, which contribute tothe interaction with target substances. Such contribution might be thedirect interaction with the target substances, or the influence on otherdirectly interacting residues, e.g. by stabilizing the conformation ofthe (poly)peptide of said unit (or module) to allow or enhance theinteraction of said directly interacting residues with said target. Suchframework and target interaction residues may be identified by analysisof the structural data obtained by the physicochemical methods referredto above, or by comparison with known and related structural informationwell known to practitioners in structural biology and/or bioinformatics.Such frameworks can also be referred to as scaffolds as they providesupport for the presentation of the more divergent target interactionresidues or CDRs.

CDRs or target interaction residues can be grafted into suitableframeworks, such as alternative scaffolds which are well-known in theart and include, but are not limited to, CTLA-4, tendamistat,fibronectin (FN3), neocarzinostatin, CBM4-2, lipocalins, T-cellreceptor, Protein A domain (protein Z), Im9, designed ankyrin-repeatproteins (DARPins), designed TPR proteins, zinc finger, pVIII, avianpancreatic polypeptide, GCN4, WW domain, Src homology domain 3 (SH3),Src homology domain 2 (SH2), PDZ domains, TEM-1 β-lactamase, GFP,thioredoxin, staphylococcal nuclease, PHD-finger, CI-2, BPT1 APPI,HPSTI, ecotin, LACI-DI, LDTI, MTI-II, scorpion toxins, insect defensin Apeptide, EETI-II, Min-23, CBD, PBP, cytochrome b₅₆₂, Ldl receptor domainA, γ-crystallin, ubiquitin, transferring, and C-type lectin-like domain(see Binz et al. (2005 October) Nat Biotech 23(10):1257-68), or intosuitable frameworks of immunoglobulin-derived antigen-bindingpolypeptides which are well-known in the art and include, but are notlimited to VhH domains, V-NAR domains, Vh domains, Fab, scFv, Bis-scFv,Camel IG, IfNAR, IgG, Fab2, Fab3, minibody, diabodies, triabodies andtetrabodies (see Holliger, P. and Hudson, P. (2005), Nat. Biotechnol.23(9), pp. 1126-1136).

Preferably, the antibody of the present invention has one or more of thefollowing further features:

-   -   it is stable under reducing conditions as measured in a yeast        interaction assay, wherein the activity of a selectable marker        protein fused to said scFv correlates with a high stability and        solubility of said scFv in an intracellular environment. Said        yeast interaction assay, the so-called “Quality control”, was        described in detail (Auf der Maur et al. (2001); Auf der Maur et        al., 2004; the references being incorporated in their entirety        herein).    -   is stable for at least 1 month, preferably at least two months,        most preferred at least six months at 20° C. to 40° C.,        preferably at 37° C. in PBS,    -   it remains monomeric under physiological conditions,    -   it is soluble at ambient temperature in PBS at concentrations        of >about 1 mg/ml, preferably of >about 4 mg/ml, more preferably        of >about 10 mg/ml, even more preferably of >about 25 mg/ml and        most preferably of >about 50 mg/ml,    -   it reveals a midpoint of transition in a guanidinium        hydrochloride titration of at least 1.5 M, preferably of at        least 1.75 M, more preferably of at least 1.9 M, most preferably        of at least 2 M, i.e. is resistant to denaturation.

The term antibody as used in the scope of the present invention refersto an scFv antibody or an antibody fragment that binds a selectedantigen. Thus, the scFv antibody of the present invention can be eithera full scFv comprising a VL and a VH domain which are linked by a shortlinker peptide, for example a linker comprising 1 to 4 repeats of thesequence GGGGS, preferably a (GGGGS)₄ peptide (SEQ ID No. 16), morepreferably a linker of the sequence GGGGSGGGGSGGGGSSGGGS (SEQ ID No:17),or a linker as disclosed in Alfthan et al. (1995) Protein Eng.8:725-731, or simply a VL or a VH domain, which has sufficient bindingcapacity for the selected antigen. The linkage of VL and VH can be ineither orientation, VL-linker-VH or VH-linker-VL.

In one aspect the present invention provides an antibody which is stablefor at least 1 month, preferably at least two months, in phosphatebuffered saline (PBS). Preferably said antibody is tested for stabilityat physiological temperature, i.e. at 37° C. In another preferredembodiment said antibody is stable for at least 6 month when kept at 4°C. in PBS, or after lyophilization at room temperature. The stabilitycan be tested for example by analyzing standard amounts of saidantibodies by SDS polyacrylamide gel electrophoresis (PAGE) followed bya standard staining procedure, such a Coomassie staining or silverstaining, and comparing the staining intensity of the full-length bandwith that of a standard protein. In addition, the absence of degradationproducts is checked. Degraded protein runs as a smear, or is eveninvisible due to the shortness of the degradation products, in whichcase only the loss of intensity of the full-length protein bandindicates degradation. In general, a physical stability of an antibodycan be assumed if no signs of aggregation, precipitation and/ordenaturation upon visual inspection of color and/or clarity or asmeasured by UV light scattering or by size exclusion chromatography areobserved.

The stability in terms of activity after a certain time of storage is afurther important feature of the antibody of the present invention. Itcan be determined comparing the potency of the antibody before and afterstorage, for example in by in vitro target binding assays using ELISA orin vivo in cellular activity assays where the inhibition potency of theantibody is measured.

In another aspect the present invention provides an antibody which isand remains monomeric under physiological conditions, as can be judgede.g. by gel filtration. The monomeric state is an important feature ofantibodies that are able to penetrate through epithelial barriers.

In a further aspect the present invention provides an antibody, which issoluble at ambient temperature in PBS at concentrations of greater thanabout 1 mg/ml, preferably of greater than about 4 mg/ml, most preferablyof about 10 mg/ml. The solubility of the purified antibody can bedetermined by PEG precipitation using PEG3000, or byself-interaction-chromatography (SIC)

In yet another aspect the present invention provides an antibody, whichreveals a midpoint of transition in a guanidinium hydrochloridetitration of at least about 1.5 M, preferably of at least about 1.75 M,more preferably of at least about 1.9 M, most preferably of at leastabout 2 M. This is a measure for stability in the sense of resistancetowards unfolding, whereby the unfolding/denaturation induced by theaddition of guanidinium hydrochloride is followed by fluoresces orcircular dichroism spectroscopy.

In a further aspect of the present invention, the antibody having one ormore of the afore mentioned biophysical characteristics is structurallycharacterized by a framework of the light chain variable domain (VL) ofat least 85% similarity, preferably at least about 95% similarity, mostpreferably at least about 98% identity to a VL framework selected fromthe group comprising SEQ. ID. NO. 1 (kappa1 type), SEQ. ID. NO. 2(kappa1 type), SEQ. ID. NO. 3 (kappa3 type) or SEQ. ID. NO. 4 (lambda1type), SEQ ID No. 5 (kappa3 type), SEQ ID No. 6 (lambda3 type), or SEQID No. 7 (lambda3 type) and/or a framework of the heavy chain variabledomain (VH) of at least 85% similarity, preferably at least about 95%similarity, most preferred at least about 98% identity to a VH frameworkselected from the group comprising Seq. Id. No. 8 (H3 type, SEQ ID No:9(H3 type), SEQ ID No. 10 (H1b type), or SEQ ID No. 11 (H3 type). In apreferred embodiment, the combination between VL homologues of SEQ IDNo:2 and VH homologues of SEQ ID No:8, combination between VL homologuesof SEQ ID No:4 and VH homologues of SEQ ID No:10, or combinationsbetween homologues of anyone of the above mentioned VL sequences a VHhomologue of SEQ ID No:9 are used. More preferred are antibodieswith >90% similarity, and even more preferred with >95% similarity toSEQ ID No. 7. Most preferred are antibodies of the sequence SEQ ID No:7and/or SEQ ID No:8. It is also understood that the invention encompassesany one of the VL sequences disclosed in combination with any one of theVH sequences disclosed so long as target binding specificity ismaintained.

The percent similarity between two sequences is a measure of the extentto which protein sequences are related. The extent of similarity betweentwo sequences can be based on percent sequence identity and/orconservation. Conservation refers to changes at a specific position ofan amino acid sequence that preserve the physico-chemical properties ofthe original residue. The similarity between sequences is typicallydetermined by sequence alignment.

The similarities referred to herein are to be determined by using theBLAST programs (Basic Local Alignment Search Tools; see Altschul, S. F.,Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic localalignment search tool.” J. Mol. Biol. 215:403-410) accessible inInternet. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain similar amino acid sequencesto the protein molecules of the invention. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST can be utilized as described inAltschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. The percentidentity between two sequences is a function of the number of identicalpositions shared by the sequences, taking into account the number ofgaps, and the length of each gap, which need to be introduced foroptimal alignment of the two sequences. The comparison of sequences anddetermination of percent identity between two sequences can beaccomplished using a mathematical algorithm, which is well known tothose skilled in the art.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package, using a NWSgapdna.CMPmatrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide oramino acid sequences can also be determined using the algorithm of E.Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has beenincorporated into the ALIGN program (version 2.0), using a PAM120 weightresi-due table, a gap length penalty of 12 and a gap penalty of 4. Inaddition, the percent identity between two amino acid sequences can bedetermined using the Needle-man and Wunsch (J. Mol. Biol. (48):444-453(1970)) algo-rithm which has been incorporated into the GAP program inthe GCG software package, using either a Blossum 62 ma-trix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

In another aspect the antibody of the present invention is chemicallymodified. Chemical modifications may change properties of the antibodysuch as stability, solubility, antigen-binding specificity or affinity,in vivo half life cytotoxicity, and tissue penetration ability. Chemicalmodifications are well known to the skilled person. A preferred chemicalmodification of the antibody of the present invention is PEGylation.

In another preferred aspect, the affinity of the antibody of the presentinvention is characterized by a dissociation constant Kd of less thanabout 100 nM, preferably less than about 10 nM, and most preferably lessthan about 1 nM. Binding parameters such as affinity of the antibody toits cognate antigen are determined by surface plasmon resonance(BiaCore) or ELISA. These methods are well known in the art.

Preferably, the antigen that is bound by the antibody of the presentinvention is TNFα (tumour necrosis factor alpha). TNFα, also known ascachectin, is a naturally occurring mammalian cytokine produced bynumerous cell types, including monocytes and macrophages in response toendotoxin or other stimuli. TNFα is a major mediator of inflammatory,immunological, and pathophysiological reactions (Grell, M., et al.(1995) Cell, 83: 793-802). A large number of disorders are associatedwith elevated levels of TNFα, many of them of significant medicalimportance. TNFα has been shown to be up-regulated in a number of humandiseases, including chronic diseases such as rheumatoid arthritis (RA),inflammatory bowel disorders including Crohn's disease and ulcerativecolitis, sepsis, congestive heart failure, asthma bronchiale andmultiple sclerosis. TNFα is also referred to as a pro-inflammatorycytokine. However, it is also involved in disorders with a localmanifestation, such as eye diseases, e.g. macular degeneration, uveitis,glaucoma, cataract, retinitis, dry eye syndrome, scleritis,conjunctivitis, and keratitis. The antibody of the present invention isparticularly suitable for the treatment of such diseases, as it can be aapplied locally and topically, for example for eye diseases by eyedrops.

The present invention also provides a DNA sequence encoding the antibodyof the present invention, as well as a cloning or expression vectorcontaining said DNA sequence. In addition, a suitable host celltransformed with said DNA sequence is provided. This can be aprokaryotic or eukaryotic cell, in particular an E. coli, yeast, plant,insect or a mammalian cell.

The antibody of the present invention may be generated using routinetechniques in the field of recombinant genetics. Knowing the sequencesof the polypeptides, the cDNAs encoding them can be generated by genesynthesis.

Furthermore, a method for the production of the antibody of the presentinvention is provided, comprising culturing of the host cell transformedwith the DNA encoding said antibody under conditions that allow thesynthesis of said antibody, and recovering said molecule from saidculture. Preferably, said method provides an scFv antibody purified fromE. coli inclusion bodies or from the E. coli periplasm, if the scFvconstruct used comprises a signal sequence that directs the polypeptideto the periplasm.

Another aspect of the present invention is the use of the antibodyprovided by the present invention as a tool for diagnostics, preferablyin vitro diagnostics, and/or as a pharmaceutical. This use isparticularly preferred in the context of any TNFα related condition. Thedisease to be treated with an anti-TNFα scFv or fragment thereof ispreferably a disease related to overexpression of TNFα. Ifoverexpression of TNFα leads to an abnormal cellular function, anantibody that can bind and thus neutralize excess TNFα is an idealpharmaceutical for the treatment of such disease, if said antibody canreach the place of TNFα excess. If this place is inside the cell, theantibody must be able to enter the cell. If this place is extracellular,the antibody must be able to reach the extracellular matrix in a tissue,i.e. it must cross at least the outermost cell layer of a tissue, whichis usually the epithelial cell layer. In another embodiment of thepresent invention the antibody is able to penetrate the endothelium.

IV. Pharmaceutical Compositions and Pharmaceutical Administration A.Compositions and Administration

In most cases the antibody of the present invention will be used in apharmaceutical composition, said pharmaceutical composition comprisingat least one further compound. Preferably, this will be in combinationwith a pharmaceutically acceptable carrier, diluent or excipient. Theexcipient may be selected from the group comprising azone, benzalkoniumchloride (BzCl), BL-7, BL-9, Brij 35, Brij 78, Brij 98, Brij 99,Polyoxyethylene-Polyoxypropylene 1800, sodium caprate, caprylic acid,cetylpyridinium chloride, chlorhexidine, cholate, castor oil, corn oil,cremophor-EL, DMSO, decamethonium bromide, deoxycholate, dextransulfate,EDTA, disodium EDETATE, ethanol, fusidate, glycocholate, lauryl sulfate,L-α-lysophosphatidylocholine, N-lauroylsarcosine, NMP, oleic acid,phospholipids, poly oxyethylene-9-lauryl ether, saponin, Tween 20, Tween40, Tween 60, Tween 80, taurocholeate, taurodeoxycholate, tight junctionopening peptides and peptide derivatives, tight junction openingproteins and protein derivatives. Preferably, the excipient is selectedfrom the group comprising benzalkonium chloride, Tween 20, Tween 40,Tween 60, Tween 80, and chlorhexidine. It is also possible to usecaprate. Such substances can work as enhancers of penetration.

In most cases the pharmaceutical composition comprising the antibody ofthe present invention will be applied locally rather than systemically.The antibody of the present invention is particularly suitable for localapplication, as its scFv format is small, and its framework hasphysicochemical characteristics enabling it to penetrate epithelialtissue barriers. A local application is an application in a relativelyrestricted area such as it is the eye, the nasal cavity, the oralcavity, the intestinal tract, the skin, the mucosa of the mouth and theurogenital tract, e joints and joint spaces, the brain, the vertebraetc., where the application of a relatively small volume of sufficientlyconcentrated antibody is effective. On the other hand, topicalapplication is an application to the surface of a body part.

The preferred form of administration of the pharmaceutical compositionof the present invention is by topical application; however, other formsare by inhalation, e.g. if the antibody is destined to penetrate thelung epithelium. Pulmonary delivery may be accomplished using an inhaleror nebulizer and a formulation comprising an aerosolizing agent.

For topical applications, the preferred locus is the eye. The antibodyof the present invention is particularly suitable to penetrate thecornea, which mainly consists of three tissue layers, namely theepithelium, the stroma and the endothelium. Thus the antibody can beused for the treatment of many diseases of the eye.

B. Drug Delivery Systems

Non-limiting examples of topical ocular drug delivery systems for use inthe invention include penetration enhancers, corneal collagen shields,ocular iontophoresis, microparticles or nanoparticles, ocular inserts,mucoadhesive polymers, in situ gelling system, dendrimers, lipidemulsions, and ocular inserts (see Sultana, et al. (2007) Future Drugs,2(2), 309-323 (2007). i.

i. Penetration Enhancers

The pharmaceutical compositions of the invention may include apenetration enhancer. Examples of penetration enhancers are well-knownin the art, and include Azone®, benzalkonium chloride (BzCl), BL-7,BL-9, Brij 35, Brij 78, Brij 98, Brij 99,Polyoxyethylene-Polyoxypropylene 1800, sodium caprate, caprylic acid,cetylpyridinium chloride, chlorhexidine, cholate, castor oil, corn oil,cremophor-EL, cyclodextrins, DMSO, decamethonium bromide, deoxycholate,dextransulfate, EDTA, disodium EDETATE, ethanol, fusidate, glycocholate,lauryl sulfate, L-α-lysophosphatidylocholine, methazolamide,N-lauroylsarcosine, NMP, oleic acid, Pz-peptide, phospholipids, polyoxyethylene-9-lauryl ether, saponin, Tween 20, Tween 40, Tween 60, Tween80, taurocholeate, and taurodeoxycholate (see Sultana, et al. (2007)Future Drugs, 2(2), 309-323 (2007). In another embodiment, thepenetration enhancer is sodium caprate. In yet another embodiment, thepenetration enhancer includes colloidal systems, polyacrylates andbio-adhesive polymer. See also U.S. Ser. No. ______ (Attorney Docket No.ES5-016-1 entitled “Improved penetration Enhancers for the Delivery ofTherapeutic Proteins” filed on Jul. 10, 2007).

ii. Corneal Collagen Shields

The antibodies of the invention can also be administered with a cornealcollagen shield. In some embodiments, collagen shields with dissolutiontimes of 12, 24 and 72 hours may be used. In other embodiments, thecollagen shield is pre-soaked in solutions of gatifloxacin and/ormoxifloxacin.

iii. Ocular Iontophoresis

The antibodies of the invention can also be delivered by oculariontophoresis. In one embodiment, the antibodies may be delivered to theanterior segment of the eye by transcorneal iontophoresis. In anotherembodiment, the high and sustained concentrations of the antibodies ofthe invention may be delivered to the vitreous and retina bytransscleral iontophoresis. Iontophoresis is applied for the desiredduration of time. In some embodiments, iontophoresis is applied about 1to about 4 minutes. In other embodiments, iontophoresis is applied lessthan 1 minute. In another embodiment, iontophoresis is applied for morethan 4 minutes.

iv. Microparticles and Nanoparticles

The antibodies of the invention can also be delivered usingmicroparticulate or nanoparticulate delivery systems. In someembodiments, the microparticle is a microcapsule. In other embodiments,the microparticle is a microsphere. In further embodiments, themicroparticulate comprises polymers which are erodible, biodegradable,nonerodible, or ion exchange resins. In another embodiment, themicroparticulate delivery system is Betoptic S®, containing betaxolol0.25%.

Nanoparticles, particles smaller than 1 μm, can also be used. In oneembodiment, the nanoparticle is a nanocapsule. In another embodiment,the nanoparticle is a nanosphere. In one embodiment, the nanoparticlecomprises polyacrylcyanoacrylate (PACA). In another embodiment, thenanoparticle comprises poly-å-caprolactone. In certain embodiments, thenanoparticle comprises solid lipid nanoparticles containing 2.5%tobramycin with hexadecyl phosphate. In another embodiment, thenanoparticle comprises Eudragit RS 100 or Eudragit RL 100 and optionallyfurther comprises cloricromene. In further embodiments, the nanoparticleincludes flurbiprofen (FB)-loaded acrylate polymer nanosuspensions.

v. Ocular Inserts

The antibodies of the invention can also be administered using ocularinserts. In some embodiments, the ocular inserts are insoluble, solubleor bioerodible inserts. The insoluble inserts include diffusionalsystems, osmotic systems and hydrophilic contact lenses. The solubleinserts can be composed of natural, synthetic, or semisyntheticpolymers. The bioerodible inserts can be composed of bioerodiblepolymers.

vi. Mucoadhesive Polymers

The pharmaceutical compositions of the invention may includemucoadhesive polymers. Examples of mucoadhesive polymers are well-knownin the art, and include chitosan (CS), Ch-HCL andN-carboxymethylchitosan (CMCh), N-trimethyl chitosan (TMC) polymers,pilocarpine-loaded CS/Carbopol®, polyacrylic acid (PAA),polysaccharides, xyloglucan, tamarind seed polysaccharide (TSP), andthiolated polymers or thiomers.

vii. In situ Gelling Systems

The pharmaceutical compositions of the invention may include in situgelling systems. Examples of in situ gelling systems are well-known inthe art, and include pH-mediated in situ gelling systems,temperature-mediated in situ gelling systems and ion-mediated in situgelling systems. pH-mediated in situ gelling systems can include, forexample, polymers such as cellulose acetate phthalate (CAP) andCarbopol®. Temperature-mediated in situ gelling systems can include, forexample, pluronics, tetraonics and ethyl hydroxyethyl cellulose. In situgelling systems may also include Gelrite® (deacetylated gellan gum(DCG)), alginates, e.g., alginate-poly(L-Lysine), timolol maleateophthalmic solutions, e.g., Timoptol XE and Lizmon TG®, and combinationsthereof.

viii. Dendrimers

The pharmaceutical compositions of the invention may include dendrimers.Examples of dendrimers are well-known in the art, and include TMpolyamidoamine (PAMAM).

C. Formulations

In another embodiment, the formulation may be a slow, extended, or timerelease formulation, a carrier formulation such as microspheres,microcapsules, liposomes, etc., as known to one skilled in the art. Anyof the above-mentioned delivery systems may be administered topically,intraocularly, subconjunctivally, or by implant to result in sustainedrelease of the agent over a period of time. The formulation may be inthe form of a vehicle, such as a micro- or macro-capsule or matrix ofbiocompatible polymers such as polycaprolactone, polyglycolic acid,polylactic acid, polyanhydrides, polylactide-co-glycolides, polyaminoacids, polyethylene oxide, acrylic terminated polyethylene oxide,polyamides, polyethylenes, polyacrylonitriles, polyphosphazenes,poly(ortho esters), sucrose acetate isobutyrate (SAIB), and otherpolymers such as those disclosed in U.S. Pat. Nos. 6,667,371; 6,613,355;6,596,296; 6,413,536; 5,968,543; 4,079,038; 4,093,709; 4,131,648;4,138,344; 4,180,646; 4,304,767; 4,946,931, each of which is expresslyincorporated by reference herein in its entirety, or lipids that may beformulated as microspheres or liposomes. A microscopic or macroscopicformulation may be administered topically or through a needle, or may beimplanted. Delayed or extended release properties may be providedthrough various formulations of the vehicle (coated or uncoatedmicrosphere, coated or uncoated capsule, lipid or polymer components,unilamellar or multilamellar structure, and combinations of the above,etc.). The formulation and loading of microspheres, microcapsules,liposomes, etc. and their ocular implantation are standard techniquesknown by one skilled in the art, for example, the use a ganciclovirsustained-release implant to treat cytomegalovirus retinitis, disclosedin Vitreoretinal Surgical Techniques, Peyman et al., Eds. (MartinDunitz, London 2001, chapter 45); Handbook of Pharmaceutical ControlledRelease Technology, Wise, Ed. (Marcel Dekker, New York 2000), therelevant sections of which are incorporated by reference herein in theirentirety. For example, a sustained release intraocular implant may beinserted through the pars plana for implantation in the vitreous cavity.An intraocular injection may be into the vitreous (intravitreal), orunder the conjunctiva (subconjunctival), or behind the eye(retrobulbar), or under the Capsule of Tenon (sub-Tenon), and may be ina depot form. The composition may be administered via a contact lensapplied to the exterior surface of an eye, with the compositionincorporated into the lens material (e.g., at manufacture, or containedin a lens solution). The composition may be administered via anintraocular lens (TOL) that is implanted in the eye. Implantable lensesinclude any IOL used to replace a patient's diseased lens followingcataract surgery, including but not limited to those manufactured byBausch and Lomb (Rochester N.Y.), Alcon (Fort Worth Tex.), Allergan(Irvine Calif.), and Advanced Medical Optics (Santa Ana Calif.). Seealso Degim, I. T and Celebi, N. (2007), Current Pharmaceutical Design,13, 99-117]. When the lens is implanted within the lens capsule, thecomposition provides the desired effect to the eye. Concentrationssuitable for implants (lenses and other types) and by contact lensadministration may vary, as will be appreciated by one skilled in theart. For example, an implant may be loaded with a high amount of agent,but formulated or regulated so that a required concentration within theabove-described ranges is sustainedly released (e.g., slow releaseformulation).

It has been found that antibody concentrations normally achieved withscFvs in the range of up to 1 mg/ml are not quite effective inepithelial penetration unless additional penetration enhancers are added(WO0040262). The present invention provides antibodies that are highlysoluble, so that pharmaceutical compositions comprising said antibody athigher concentrations, i.e. greater than about 2 mg/ml, preferablygreater than about 5 mg/ml, most preferably greater than about 10 mg/mlcan be prepared and used in effective treatment of the corresponding,antigen related disease.

Hence, the present invention provides a method for the treatment of anantigen-related disease, wherein delivery of the antibody to the site ofantigen-antibody interaction requires penetration of a tissue thatcomprises tight junctions, in particular an epithelium and/or anendothelium. An epithelium is a tissue composed of a layer of cells, andit lines both the outside (skin) and the inside (e.g. intestinum) oforganisms. Epithelia also include the mucous membranes lining the insideof mouth and body cavities and comprise dead squamous epithelial cells,and epithelial cells lining the inside of the lungs, thegastrointestinal tract, and the reproductive and the urinary tracts. Anendothelium is a layer of thin, flat cells that lines the interiorsurface of blood vessels and organs. In vasculature it forms aninterface between circulating blood in the lumen and the rest of thevessel wall. Endothelial cells line the entire circulatory system, fromthe heart to the smallest capillary. In small blood vessels andcapillaries, endothelial cells are often the only cell-type present.Endothelial cells also control the passage of materials into and out ofthe bloodstream. In some organs, there are highly differentiatedendothelial cells to perform specialized ‘filtering’ functions. Examplesof such unique endothelial structures include the renal glomerulus andthe blood-brain barrier. Endothelial tissue is a specialized type ofepithelium tissue.

In a preferred embodiment of the present invention, the penetration ofthe cornea can be achieved. The corneal epithelium covers the front ofthe cornea and consists of several layers of cells, which renderspenetration by an antibody even more difficult. The antibody of thepresent invention is preferably able to penetrate the entire cornea.

An ideal drug for treatment of uveitis should cover four crucialcharacteristics: 1) Provide a fast onset of effects on acute symptoms.2) Show comparable efficacy as compared with standard topicalcorticosteroids. 3) Show superior safety profile as compared withstandard topical corticosteroids. 4) Have favourable pharmacokineticproperties allowing for topical application to the cornea as well as forintravitreal injection as compared with standard corticosteroids orstandard monoclonal antibodies.

A locally applied inhibitor of TNFα, comprising adequate pharmacokineticproperties can address all these aspects. The good penetration of theanti-TNFα scFv antibody ESBA105 into the anterior part of the eyecombined with its high TNFα-neutralizing activity ultimately lead tointra-ocular concentrations, which are very well suitable fortherapeutic efficacy for uveitis anterior. Taken into account that TNFαlevels found in the diseased eye were 15 pg/ml and that ESBA105concentrations measured were up to 40,000 ng/ml for in the anterior part(see FIG. 7) and up to 125 ng/ml for vitreous part of the eye (see FIG.8), we conclude that ESBA105 is not only suitable for the treatment ofuveitis anterior, but also for the treatment of different forms ofpanuveitis like e.g. ocular Behçet's disease. The present invention thusprovides an antibody for the treatment of ocular disorders by topicalapplication to the eye, in particular for the treatment of any form ofuveitis, Behçet's disease, retinitis, dry eye syndrome, glaucoma,Sjögren syndrome, diabetes mellitus (incl. diabetic neuropathy),scleritis, conjunctivitis and keratitis. Administration preferablyoccurs by eye drops, eye ointment, or from depot devices like contactlenses).

In another preferred embodiment of the present invention the antibodymust penetrate the intestinal epithelium.

In the case of treatment in the lung, inhalation must be used to bringthe antibody to apply the antibody of the present invention at the lungepithelium.

In yet another aspect of the present invention, the antibody is used asa diagnostic tool.

The sequences of the present invention are the following:

VL of the kappa 1 type SEQ.ID. No: 1EIVMTQSPSTLSASVGDRVIITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQYKSYWTFG QGTKLTVLGVL of the kappal type SEQ ID No: 2EIVLTQSPSSLSASVGDRVTLTCRASQGIRNELAWYQQRPGKAPKRLIYAGSILQSGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQYYSLPYMF GQGTKVDIKRVL of the kappa3 type SEQ ID No: 3EIVMTQSPATLSVSPGESAALSCRASQGVSTNVAWYQQKPGQAPRLLIYGATTRASGVPARFSGSGSGTEFTLTINSLQSEDFAAYYCQQYKHWPPWT FGQGTKVEIKRVL of the kappa3 type SEQ ID No: 4 EIVLTQSPATLSLSPGERATLSCRASQTLTHYLAWYQQKPGQAPRLLIYDTSKRATGTPARFSGSGSGTDFTLTISSLEPEDSALYYCQQRNSWPHTF GGGTKLEIKRVL of the lambda1 type SEQ ID No: 5 QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLIYDNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSG VVFGGGTKLTVLGVL of the lambda 3 type SEQ ID No: 6SYVLTQPPSVSVAPGQTATVTCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTIRRVEAGDEADYYCQVWDSSSDHNV FGSGTKVEIKRVL of the lambda 3 type SEQ ID No: 7 LPVLTQPPSVSVAPGQTARISCGGNNIETISVHWYQQKPGQAPVLVVSDDSVRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDYVV FGGGTKLTVLGVH of the H 1b type SEQ ID No: 8 QVQLVQSGAEVKKPGASVKVSCTASGYSFTGYFLHWVRQAPGQGLEWMGRINPDSGDTIYAQKFQDRVTLTRDTSIGTVYMELTSLTSDDTAVYYCARVPRGTYLDPWDYFDYWGQGTLVTVSS VH of the H3 type SEQ ID No: 9 QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAHVLRFLEWLPDAFDIWGQGTLVTVSS VH of the H3 type SEQ ID No: 10 EVQLVESGGGVAQPGGSLRVSCAASGFSFSSYAMQWVRQAPGKGLEWVAVISNDGRIEHYADAVRGRFTISRDNSQNTVFLQMNSLRSDDTALYYCAR EIGATGYLDNWGQGTLVTVSSVH of the H3 type SEQ ID No: 11 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDAGIAVAGTCFDYWGQGTLVTVSS TB-A SEQ ID No: 12 DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS ESBA105 SEQ ID No: 13 DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSSGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS TB-WT SEQ ID No: 14 DIVMTQTPKFLLVSAGDRVTITCTASQSVSNDVVWYQQKPGQSPKMLMYSAFNRYTGVPDRFTGRGYGTDFTFTISSVQAEDLAVYFCQQDYNSPRTFGGGTKLEIKRGGGGSGGGGSGGGGSSGGGSQIQLVQSGPELKKPGETVKISCKASGYTFTHYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKEHFAFSLETSASTVFLQINNLKNEDTATYFCARERGDAMDYWGQGTSVTVSS scFv LucentisSEQ ID No: 15  DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRGGGGSGGGGSGGGGSSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYTSSHWYFDVWGQGT LVTVSS linkerSEQ ID No: 16 GGGGSGGGGSGGGGSGGGGS linker SEQ ID NO: 17 GGGGSGGGGSGGGGSSGGGS ESBA105-QC15.2 SEQ ID No: 18 DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSSGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGRGLEWMGWINTYTGEPTYADKFKDRITFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS ESBA105-H_F68ASEQ ID No: 19  DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSSGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRATFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS TB-H_F68V_F70LSEQ ID No: 20  DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSSGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRVTLSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS TB-H_F70LSEQ ID No: 21 DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSSGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTLSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS

EXEMPLIFICATION

Throughout the examples, the following materials and methods were usedunless otherwise stated.

Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, immunology (especially, e.g.,immunoglobulin technology), and animal husbandry. See, e.g., Sambrook,Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor LaboratoryPress (1989); Antibody Engineering Protocols (Methods in MolecularBiology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: APractical Approach (Practical Approach Series, 169), McCafferty, Ed.,Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., C.S.H.L.Press, Pub. (1999); Current Protocols in Molecular Biology, eds. Ausubelet al., John Wiley & Sons (1992)

Example 1 Production of scFvs

ScFv antibodies were produced via expression as inclusion bodies,followed by a refolding and a chromatography step. Three single-chainantibodies were produced. This includes two conventional scFvs, whichpresumably do not fulfill the criteria of being well soluble,particularly stable and monomeric. One of them is TB-wt (SEQ ID No:14),which was constructed by linking the natural VL and VH sequences of themurine anti-TNFα monoclonal antibody Di62 (Doring et al., 1994). Theother one is lucentis-scFv, which was constructed by linking the VL andVH sequences of the VEGF-specific Fab fragment ranibizumab (SEQ. ID.No:15). ESBA105 is an scFv, which carries the CDRs of Di62 and whichreveals a Kd value in the nanomolar range for TNFα. Solubility,stability and monomeric behavior of ESBA105 were optimized by employingan scFv framework, which was selected by the so-called Quality Controlsystem (Auf der Maur et al., 2004). The amino acid sequence of ESBA105is disclosed in SEQ ID No. 13.

FIG. 1 shows the elution profiles of ESBA105 (A), TB-wt (B) andlucentis-scFv (C) of the preparative gel filtration, which follows therefolding step. Whereas the profile for ESBA105 consists mainly of onesharp peak going up to 300 mAU (relative units for absorption), severalbroad peaks with maximal highs of 16 and 55 mAU, respectively, can beseen in the profiles of TB-wt and lucentis-scFv. This indicates thateither the quality of the inclusion bodies and or the refolding processis much less efficient for TB-wt and lucentis-scFv as compared toESBA105.

Below, the production procedure is described in detail for ESBA105.

Construction of Plasmid pGMP002 for Expression of ESBA105:

The ESBA105 coding sequence was ligated into the NcoI-HindIII sites ofpET-24d(+) (Novagen, catalogue number 69752-3). Subsequently, theBpu11021-NotI fragment of the resulting construct was removed by bluntend ligation after the sticky ends produced by Bpu11021 and NotIdigestions were filled up by T4 polymerase reaction to produce pGMP003.pGMP002 was produced by removing the Bpu11021-NotI from pET-24d(+)directly.

Expression of ESBA105 in E. coli:

For expression of ESBA105, E. coli BL21 (DE3) (Novagen) was transformedwith the expression plasmid pGMP002. Glycerol stock cultures wereprepared from single colonies, cultivated in M9 medium (Sambrook et. al,Molecular Cloning, A laboratory Manual) containing 1% glucose, 1 ml/l ofa trace element solution (Wang and Lee, Biotechnol Bioeng 58:325-328)and 50 μg/ml kanamycin. Cells were grown overnight at 37° C., glycerolwas added to 20% and 1 ml aliquots were stored as glycerol stocks at−80° C.

For the first preculture, 50 ml of a synthetic defined medium (Korz et.al., J. Biotechnol. 1995, 39:59-65), containing 1% glucose, 1 ml/l oftrace element solution and 50 μg/ml kanamycin, was inoculated with 1 mlof a glycerol stock. Preculture was grown overnight at 37° C. and 200rpm in a baffled shake flask. A second preculture with 250 ml of thesame medium was inoculated with the first preculture and grown foradditional three hours at 37° C.

Bioreactor cultivations were performed in a 5 l bioreactor (BioFlo 110,New Brunswick Scientific), containing an initial volume of 3 l of thesame synthetic medium used for precultures. Cultivation temperature wasset to 37° C. Culture pH was controlled at 7.0 by the addition of 25%ammonia water and 1 M phosphoric acid. The level of dissolved oxygen wasmaintained above 20% of the air saturation level by varying the pureoxygen percentage at an overall aeration rate of 4 l/min. The bioreactorwas inoculated with the second preculture and operated in batch mode upto an OD₆₀₀ of 10-12. Exponential glucose feeding was then started at arate of 0.15 h⁻¹ with a 50% glucose solution containing 10 g/lMgSO₄.7H₂O and 5 ml/l trace element solution. After 16 h, proteinexpression was induced by addition of IPTG to 1 mM and exponentialfeeding was continued for 3 h. Cells were then harvested bycentrifugation at 4,600 rpm for 1 h.

Inclusion Body Preparation:

For preparation of inclusion bodies, 1 kg of wet cell paste wasresuspended in 5 l TBS buffer (10 mM Tris, 150 mM NaCl, pH 7.3). 1 g ofsolid lysozyme was added and cell suspension was incubated for 30-60 mM.For cell disruption, the suspension was passed two times through ahigh-pressure homogenizer (Niro Soavi, Panda 2K) at 1,000 bar. Disruptedcells are centrifuged for 1 h at 4,600 rpm and the resulting inclusionbodies were resuspended in 21 TBS buffer, containing 0.5% LDAO(N-,N-Dimethyldodecylamin-N-oxid, Fluka). The inclusion bodies werewashed repeatedly until no significant residual protein was detected inthe washing supernatant. Finally, inclusion bodies were washed twicewith TBS-buffer without LDAO.

Refolding of ESBA105:

Inclusion bodies were solubilized in the 10-fold volume ofsolubilization buffer containing 6 M guanidinium-HCl, 100 mM Tris, 1 mMEDTA and 20 mM DTT at pH 8.0 and incubated overnight at roomtemperature. Solubilized protein was then refolded by 1:50 dilution intorefolding buffer containing 3 M urea, 100 mM Tris and 2 mM each ofcystein and cystine at pH 8.5. Refolding was continued for 24 h at roomtemperature. After refolding, precipitates were removed by depthfiltration and the refolding solution was concentrated to 50% of theinitial volume. The concentrated refolding solution was then dialyzedagainst the 4-fold volume of PBS buffer (50 mM Na-phosphate, 150 mMNaCl, pH 6.5) and further concentrated to a protein concentration ofapproximately 1 mg/ml.

Purification of ESBA105 by Chromatography:

ESBA105 was purified in two chromatography steps: gel filtration andcation exchange chromatography. All chromatography steps were operatedby ÄKTA purification systems (GE Healthcare).

For preparative gel filtration chromatography, a Sephadex S75 26/60column (GE Healthcare) was used with PBS buffer (50 mM Na-phosphate, 150mM NaCl, pH 6.5) as running buffer. Collected ESBA105-fraction waseluted at approximately 185 ml retention volume as a single peak.

Fractogel EMD SO₃ ⁻ (M) (Merck) was used as column resin for the secondchromatography step. Resin was equilibrated with five column volumesequilibration buffer (50 mM Na-acetate, pH 5.5). ESBA105 peak fractionsfrom gel filtration were diluted 10 fold with 50 mM Na-acetate pH 5.5and then loaded onto the column with a flow rate of 5 ml/min. Afterloading, the column was washed with five column volumes equilibrationbuffer. A linear gradient from 0-35% Elution buffer (50 mM Na-acetate,500 mM NaCl, pH 5.5) within 60 minutes and 5 ml/min was used for elutionof ESBA105. The most prominent peak after elution was collected andidentified as ESBA105. The collected ESBA105 fraction was finallydialyzed against PBS (50 mM Na-phosphate, 150 mM NaCl, pH 6.5) andstored at −80° C.

Example 2 Biophysical Characterization of scFvs

In order to investigate their “drug-likeness”, ESBA105 and some of itsderivatives were biophysically characterized. Characterization involvedthe determination of (1) solubility parameters (PEG precipitation andB₂₂ value; the B22 value is a measure for protein self-interaction,which is important in protein crystal growth, solubilisation andaggregation. See Valente et al., Biophys J. 2005 December; 89(6):4211-8.Epub 2005 Sep. 30), (2) the pI values, (3) the measurement of thermaland chaotropic denaturation, and (4) the quantification of the monomericfraction as compared the oligomeric fraction. The results of items 1-3are summarized in Table 1, which also includes relative potency valuesas determined by the L929 and the KYM-1 assay, respectively.

TABLE 1 Summary of data on some ESBA105 derivatives. S_(max) SEQ I (PEGprec.) B₂₂ value (SIC) ESBA105 7.8 1.839 ± 0.133 −24.5 × 10⁻⁴ ± 3.8 ×10⁻⁴   ESBA105-QC2.2 7.8 nd nd ESBA105-QC7.1 nd nd ESBA105-QC11.2 8.21.913 ± 0.091 1.59 × 10⁻³ ± 5.9 × 10⁻⁵ ESBA105-QC15.2 7.8 1.857 ± 0.02 1.28 × 10⁻³ ± 3.0 × 10⁻⁴ ESBA105-QC23.2 7.8 1.881 ± 0.068 1.06 × 10⁻⁴ ±2.9 × 10⁻⁵ ESBA105-H_F68A 7.8 nd nd TB-H_F68V_F70L 7.8 nd nd TB-H_F70L7.8 nd nd Onset Relative potency: of EC₅₀X/ dena- [GdnHCl] atEC₅₀ESBA105 turation midpoint of L929 KYM-1 SEQ [° C.] unfolding cellscells ESBA105 53 2.07M 1.0 1.0 ESBA105-QC2.2 58 nd 1.1 1.6 ESBA105-QC7.1nd nd nd ESBA105-QC11.2 58 2.33M 0.8 1.3 ESBA105-QC15.2 26 2.30M 1.371.5 ESBA105-QC23.2 58 nd 1.32 1.5 ESBA105-H_F68A nd nd 1.14 ndTB-H_F68V_F70L nd nd 1.28 nd TB-H_F70L nd nd 2.7 nd

Example 3 Stability of scFv During Storage

The stability of the scFv over time is an important feature in thecontext of a pharmaceutical. FIG. 4 shows an analysis of the ESBA105scFv (lanes 1-6) and the QC15.2 scFv (lanes 7-12) that were separated bySDS-PAGE and stained with Coomassie after two weeks of storage atdifferent temperature (−80° C.: lanes 1, 3, 5, 7, 9, and 11) or 40° C.:lanes 2, 4, 6, 8, 10 and 12) and concentrations (20 mg/ml: lanes 1, 2, 7and 8; 10 mg/ml; lanes 3, 4, 9 and 10; 1 mg/ml: lanes 5, 6, 11 and 12).The antibodies remained stable over the entire ranges of temperature andat all tested concentrations, and no signs of degradation or aggregationwere found.

An antibody that is stable over a certain time should also retain itsfull activity. Therefore, we tested ESBA105 eight weeks after storage at37° C. or −80° C. in L929 assays for their activity. The result ispresented in FIG. 5, and no difference between the two temperaturescould be seen.

Example 4 Ex Vivo Penetration of ESBA105 into Whole Rabbit Eyes

The amount of ESBA105 penetrating into different compartments of wholerabbit eyes was tested under various conditions in an ex vivo setting.For these series of experiments, the rabbit eyes were placed onincubation test plates that have depressions on its surface. Thedepression contained the ESBA105 test solution and the rabbit eyes wereplaced on the test plates such that the cornea was in contact with thetest solution. After an incubation time of 4 hours at 37° C., thedifferent eye compartments were analyzed for their ESBA105 content. Forthis analysis, a syringe was used to remove probes from the compartmentof interest (see FIG. 6) and the ESBA105 concentration of the probe wasdetermined by ELISA.

The following conditions were assessed with the experimental setupdescribed above: ESBA105 was tested at concentrations of 1, 2, 5 and 10mg/ml. The concentration of 1 mg/ml was furthermore tested in thepresence of 0.5% capric acid. 0.5% of capric acid was previously shownby Thiel et al. (Clin. Exp. Immunol. 2002 April; 128 (1):67-74) toenhance the penetration through cornea. When 1 mg/ml was administered inthe presence of 0.5% capric acid, we measured a concentration of ESBA105in the anterior part of the eye of about 40 μg/ml, which was about thesame as compared to concentrations measured after administration of 10mg/ml in the absence of capric acid (FIG. 7). Interestingly, when theresults of these two administration modes were compared in the vitreousliquid, there were about 40 ng/ml measured for administration of 1 mg/mlwith capric acid and about 125 ng/ml for 10 mg/ml without capric acid(FIG. 8). In the absence of capric acid the concentrations measured inthe two compartments revealed a clear dependence on the appliedconcentrations of 1, 2, 5 and 10 mg/ml (FIG. 8).

The eye incubation test plates were pre-incubated with blocking buffer(5% low fat milk powder in PBS, pH 7.4) over night at 4° C. On the nextday the plates were washed with PBS and afterwards dried by incubatingat 37° C. for 30 minutes. The wells of the dried plates were filled with125 μl of test solution each and isolated rabbit eyes were placed to thewells in a manner that only the cornea was in contact with the testsolution. After incubation at 37° C. and 100% humidity for 4 hours, theeyes were washed for 3 hours with PBS. Paracenthesis was carried outusing a 25 G needle for the anterior eye chamber and a 22 G needle forthe vitreous body.

The concentration of ESBA105 in the probes was determined by ELISA asfollows: The probes were centrifuged with a bench-microfuge at 13,000rpm for 5 min at room temperature. The probes from the anteriorcompartment were diluted 1:10, 1:100 and 1:1000, the probes from thevitreous compartment were diluted 1:5 in TBST (TBS supplemented with0.005% Tween) containing 0.5% low fat milk powder (hereinafter referredto as diluting solution) for ELISA testing.

ELISA 96 well plates (Nunc Maxisorb; catalogue number 442404) werecoated with 0.5 μg/ml human TNFα over night at 4° C. On the next day theplates were washed 3 times with TBST at room temperature and incubatedwith 300 μl blocking buffer (5% low fat milk powder in TBST) per wellfor 60 min at room temperature on a shaker (500-600 rpm). After theblocking step the plates were washed 3 times with TBST before 50 μl ofdiluted probes were added to each well and the plates were incubated ona shaker (500-600 rpm) for 90 minutes at room temperature. After thisincubation step the plates were washed 3 times with TBST, before 50 μlof a 1:10,000 solution (diluted in diluting solution) of the secondaryantibody AKA3A-biotinylated were added to each well. AKA3A-biotinylatedis a polyclonal rabbit anti-ESBA105 antibody, which was freshlybiotinylated (according to standard biotinylation protocols). After 90minutes of incubation on a shaker (500-600 rpm) at room temperature, theplates were washed 3 times with TBST, before 50 μl of a 1:2000 dilution(diluted in diluting solution) of streptavidin-coupled horseradishperoxidase (streptavidin-polyHRP40, 1 mg/ml; SDT; #SP40C) was added.After 60 minutes of incubation on a shaker (500-600 rpm) at roomtemperature, the plates were washed 3 times with TBST and 2 times withddH₂O. Horseradish peroxidase (HRP) activity was detected upon additionof 50 μl BM Blue POD substrate (Roche Diagnostics, catalogue number1484281) to each well. After 6-12 minutes of incubation at roomtemperature in the dark the HPR reaction was stopped by addition of 50μl 1 M HCl to each well. The HPR reaction was quantified byspectrometric measurement at 450 nm in a TECAN Genios reader.

The ESBA105 concentration of the probes was finally determined bycomparison to a standard curve, which was produced in parallel.

Example 5 Penetration Through Caco-2 Cell Monolayers by ESBA105

Human colon adenocarcinoma (Caco-2) cells were seeded on permeablesupports of a 24-transwell plate and cultivated during 21 days at 37° C.in a 5% CO₂ atmosphere to allow the formation of tight monolayers.Before performing the permeability assay the transepithelial electricalresistance was measured in each well to verify the integrity of themonolayers. Caco-2 monolayers were then washed three times with a salinesolution (HBSS) and a mixture containing either 1 μM of ESBA105 with orwithout caprate, or assay medium alone was added to the uppercompartment of the transwells as a control. After defined time pointsthe content of the lower compartments was collected and replaced byfresh assay medium. Samples collected were subsequently analyzed byquantitative ELISA to determine the amount of ESBA105 that penetratedthrough the Caco-2 epithelial monolayer (FIG. 9).

Example 6 Penetration Through Mouse Jejunum

Approximately 5 cm of mouse jejunum were excised immediately afteranimal euthanasia and were incubated in oxygen saturated ringer-krebsbuffer and flushed three times with ringer-krebs buffer. A section of3.5 cm was ligated with surgical silk and filled with 200 mcl antibodymixture, containing 1 mg/ml of each antibody format (Infliximab andESBA105). Approximately 1 cm distal of the everted sac compartment asecond ligation was added to secure tightness of the compartment.Everted sacs were placed into a beaker glass containing 10 ml of oxygensaturated ringer-krebs buffer such that only first ligations came intouch with the buffer. Protease inhibitors were added to this receptorcompartment to prevent degradation of antibodies. In order to determineamount of each antibody that penetrated through intestinal tissue, 200mcl probes were taken from the receptor compartment after defined timepoints and were subsequently analyzed by quantitative ELISA.

Example 7 Topical Application of a Soluble Antigen-Binding Polypeptide

The pharmacodynamics and pharmacokinetics of the topical application ofa soluble antigen-binding polypeptide, ESBA105, in rabbit eyes in vivowere studied. Four different formulations were prepared. The firstformulation (referred to herein as “B15”) was comprised of 9.6 mg/ml ofESBA105 in 0.15M phosphate buffer at pH 6. The second formulation(referred to herein as “B16”) was comprised of 10.3 mg/ml of ESBA105 and0.01% (w/v) chlorhexidine in 0.15M sodium phosphate buffer at pH 6. Thefirst control (referred to herein as “B15-0”) was comprised of 0.15Msodium phosphate buffer at pH 6. The second control (referred to hereinas “B 16-0”) was comprised of 0.01% (w/v) chlorhexidine in 0.15M sodiumphosphate buffer at pH 6. All formulations were sterilized prior to use,and stored at 4° C. As used herein, “OTT” indicates no treatment.

To study the pharmacodynamics of ESBA105 in the eye, 6 rabbits were usedfor each formulation. The formulation was tested in both eyes of therabbits. Two rabbits (4 eyes) were used for each formulation withoutantibody fragments, and two rabbits (4 eyes) were used as naïvecontrols. A total of 18 rabbits were used for this study.

The formulations were applied locally to the eyes over the course of sixdays. Rabbits were treated five times a day, at hour seven, hour ten,hour thirteen, hour seventeen, and hour twenty. On the day of sacrifice,rabbits were treated twice, at hour seven, and hour ten, and sacrificedone hour after the last application.

The eyes were evaluated at day one, day three, and day six. Two rabbitsper formulation were used to gather pharmacokinetic data for each timepoint. An aqueous humour sample and a serum sample were taken at day oneand day three. At day six, an aqueous humour sample, and a serum samplewere taken and the eyes of the rabbits were dissected into tissues inorder to study the distribution of the antibody fragment in thedifferent eye tissues. The results of this study are graphicallydepicted in FIGS. 11 a through 11 d.

Additional studies were performed to study the solubility,pharmacodynamics and pharmacokinetics of ESBA105. The results arerecorded in FIGS. 12 through 16. As can be seen from these figures,ESBA105 has been shown to penetrate through corneal tissue layers inrabbits and pigs. ESBA105 accumulates in the vitreous and has a localhalf time of about 25 hours. The local half time of ESBA105 in theanterior chamber is significantly lower than in the vitreous. Systemicexposure upon topical treatment is extremely low, e.g., approximately 2%of highest local concentration. Further, based on the rabbit topicalexperiment therapeutic drug levels (approximately 16,000 fold about TNF)can be reached with as little as five topical drops per day. Therefore,topical application of TNF inhibitory scFv antibody fragments could bean effective therapy in diseases located in more posterior segments ofthe eye, e.g., vitreous, and retina.

Example 8 Dose Response in the Rat Acute Monoarthritis Model

To study the dose response, ESBA105 was administered as shown in FIG.17. As a comparison, Remicade® (infliximab) was administered as shown inFIG. 17. For this experiment, TNFα was administered with three differentdosages (156 μg, 45 μg, or 11 μg) of ESBA105 or infliximab. As controls,PBS, and TNF alone (10 μg i.a.), or scFv alone were used. The resultsare shown in FIG. 17.

Example 9 Topical Application In Vivo

To determine the maximal systemic exposure and local drug levels duringone day of topical administration, one drop every 20 minutes wasadministered over a period of 10 hours. Drug levels were measured byELISA in aequeous, vitreous and serum. ESBA105 was formulated as 10mg/ml in PBS, pH 6.5. For each drop, 30 mcl was applied to the top ofthe pupil, and the eye lids were then squeezed to remove excess fluid.The results are shown in FIG. 18 and Tab. 2.

TABLE 2 shows the maximal ESBA105 concentrations (Cmax) measured in theindicated compartments in the course of the experiment as described inthe legend of FIG. 18 A. The percentage values indicate the percentageof Cmax as compared to the total applied dose (cumulative dose). Cmaxcumulative total dose Vitr.  4.0 ng/ml (6.6E−5%) 9 mg Aequ.  5.3 ng/ml(1.2E−5%) 9 mg Serum 14.2 ng/ml (0.3%) 9 mg

Example 10 Topical Application of a Soluble Antigen-Binding Polypeptide

To determine the maximal systemic exposure and local drug levels duringchronic topical low frequency treatment, one drop 5 times a day wasapplied to two rabbits (4 eyes) for up to 6 days. For each drop, 30 mclof ESBA105 was applied into the lower eye sac (presentation of drugmainly to sclera), full 30 mcl remaining on ocular surface.

The eyes were evaluated after the second drop on day two, day three, andday six. Drug levels were measured by ELISA in aequeous, vitreousneuroretina, choroidea and serum. The results of this study aregraphically depicted in FIGS. 19 a through 19e.

Table 3 summarizes the median scFv ESBA105 concentrations found afterthe 6^(th) sixth day of administration in the indicated compartments in[ng/ml].

TABLE 3 Median scFv ESBA105 concentrations found after the 6^(th) sixthday of administration Aequeous Vitreous Neuroretina Choroides SerumMedian scFv ~100 ~350 ~300 ~100 <1 concentration after 6^(th) day[ng/ml]

Numerous modifications and alternative embodiments of the presentinvention will be apparent to those skilled in the art in view of theforegoing description. Accordingly, this description is to be construedas illustrative only and is for the purpose of teaching those skilled inthe art the best mode for carrying out the present invention. Details ofthe structure may vary substantially without departing from the spiritof the invention, and exclusive use of all modifications that comewithin the scope of the appended claims is reserved. It is intended thatthe present invention be limited only to the extent required by theappended claims and the applicable rules of law.

All literature and similar material cited in this application,including, patents, patent applications, articles, books, treatises,dissertations and web pages, regardless of the format of such literatureand similar materials, are expressly incorporated by reference in theirentirety. In the event that one or more of the incorporated literatureand similar materials differs from or contradicts this application,including defined terms, term usage, described techniques, or the like,this application controls.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way.

While the present inventions have been described in conjunction withvarious embodiments and examples, it is not intended that the presentteachings be limited to such embodiments or examples. On the contrary,the present inventions encompass various alternatives, modifications,and equivalents, as will be appreciated by those of skill in the art.

The claims should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail may be made without departing fromthe scope of the appended claims. Therefore, all embodiments that comewithin the scope and spirit of the following claims and equivalentsthereto are claimed.

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1. An antigen-binding polypeptide represented by the formula: Y-L-Z; orZ-L-Y; with Y being [F1-CDR1-F2-CDR2-F3-CDR3F4] and Z being[F5-CDR1-F6-CDR2-F7-CDR3F8]; wherein framework regions (F1-F4) of Y areat least 85% identical to the framework regions of the human light chainframework set forth in SEQ ID NO: 1; framework regions (F5-F6) of Z areat least 85% identical to the framework regions of the human heavy chainframework set forth in SEQ ID NO: 10; CDRs (CDRs1-3) of Y are derivedfrom one or more non-human donor CDRs capable of binding a targetantigen; CDRs (CDRs4-6) of Z are derived from one or more non-humandonor CDRs capable of binding the target antigen; and L is a flexiblepolypeptide linker.
 2. An antibody comprising: (a) a light chainvariable domain (VL) having three non-human VL CDR regions and VLframework regions of at least about 85% similarity to VL frameworkregions of a light chain variable domain selected from the groupconsisting of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7; and (b) a heavychain variable domain (VH) having three non-human VH CDRs and VHframework regions of at least about 85% similarity to VH frameworkregions of a heavy chain variable domain selected from the groupconsisting of: SEQ ID NOs: 8, 9, 10 and
 11. 3. The antibody of claim 2,comprising a light chain variable domain (VL) framework of at leastabout 95% similarity to the framework sequence SEQ ID NO: 1 and a heavychain variable domain (VH) framework of at least about 95% similarity tothe framework sequence of SEQ ID NO:
 10. 4. An isolated polynucleotidemolecule encoding the antigen-binding polypeptide of claim 1 or theantibody of claim
 2. 5. A cloning or expression vector comprising theisolated polynucleotide molecule according to claim
 4. 6. A suitablehost cell transformed with an expression vector according to claim
 5. 7.The host cell of claim 6, being a prokaryotic or eukaryotic cell, inparticular an E. coli, yeast, plant, insect or a mammalian cell.
 8. Apharmaceutical composition comprising the antigen-binding polypeptide ofclaim 1 or the antibody of claim
 2. 9. The pharmaceutical composition ofclaim 8, being formulated for topical application to mucous membranes,skin or eye.
 10. The pharmaceutical composition of claim 8, beingformulated for intramuscular, subcutaneous, intravenous, intraaterial,intrathecal or intraperitoneal injection.
 11. The pharmaceuticalcomposition of claim comprising the antigen-binding polypeptide of claim1 formulated to achieve an intraocular concentration of at least 100ng/ml or more.
 12. The pharmaceutical composition according to claim 11,formulated for topical administration to yield an intraocularconcentration of 100 ng/ml or more based on a cellular or animal modelsystem.
 13. The pharmaceutical composition according to claim 11,formulated to remain soluble at high concentrations.
 14. Thepharmaceutical composition of claim 10 formulated for topicalapplication to eye and capable of passing through the cornea and into anintraocular space in the absence of penetration enhancer.
 15. A methodfor treating an eye disease or disorder comprising administering apolypeptide of claim 1 to a patient in need thereof.